Eva Berra

Primary Mediastinal B-Cell Lymphoma: High Frequency of BCL-6 Mutations and Consistent Expression of the Transcription Factors OCT-2, BOB.1, and PU.1 in the Absence of Immunoglobulins

Although primary mediastinal (thymic) large B-cell lymphoma has been primarily studied, its precise phenotype, molecular characteristics, and histogenesis are still a matter of debate. The International Extranodal Lymphoma Study Group collected 137 such cases for extensive pathological review. Histologically, the lymphomatous growth was predominantly diffuse with fibrosis that induced compartmentalized cell aggregation. It consisted of large cells with varying degrees of nuclear polymorphism and clear to basophilic cytoplasm. On immunohistochemistry, the following phenotype was observed: CD45(+), CD20(+), CD79a(+), PAX5/BSAP(+), BOB.1(+), Oct-2(+), PU.1(+), Bcl-2(+), CD30(+), HLA-DR(+), MAL protein(+/-), Bcl-6(+/-), MUM1/IRF4(+/-), CD10(-/+), CD21(-), CD15(-), CD138(-), CD68(-), and CD3(-). Immunoglobulins were negative both at immunohistochemistry and in situ hybridization. Molecular analysis, performed in 45 cases, showed novel findings. More than half of the cases displayed BCL-6 gene mutations, which usually occurred along with functioning somatic IgV(H) gene mutations and Bcl-6 and/or MUM1/IRF4 expression. The present study supports the concept that a sizable fraction of cases of this lymphoma are from activated germinal center or postgerminal center cells. However, it differs from other aggressive B-cell lymphomas in that it shows defective immunoglobulin production despite the expression of OCT-2, BOB.1, and PU.1 transcription factors and the lack of IgV(H) gene crippling mutations.

Molecular histogenesis of plasmablastic lymphoma of the oral cavity.

Summary. Plasmablastic lymphoma (PBL) of the oral cavity is an aggressive B‐cell lymphoma associated with human immunodeficiency virus infection. Although the lymphoma phenotype is consistent with late B‐cell maturation, the molecular histogenesis of PBL is unknown. We investigated PBL of the oral cavity (n = 12) for mutations of immunoglobulin variable heavy chain (IgVH) and BCL‐6 genes, which are acquired by B cells at the time of germinal centre (GC) transit, and for expression of BCL‐6, MUM‐1 and CD138, which distinguish GC B cells from post‐GC B cells. Somatic IgVH hypermutation occurred in 4/10 PBL whereas 6/10 PBL displayed germline IgVH genes. Among PBL carrying hypermutated IgVH genes, the pattern of IgVH mutations was consistent with antigen stimulation in two cases. Mutations of the BCL‐6 gene were restricted to 1/12 patients with PBL of the oral cavity. All cases of PBL of the oral cavity displayed the BCL‐6–/MUM‐1+/CD138+ phenotype that is consistent with late stage of B‐cell differentiation. Overall, these data indicate that, despite a common phenotype and an apparently similar degree of differentiation, PBL of the oral cavity are characterized by histogenetic heterogeneity. A subset of PBL of the oral cavity carried the molecular clues of GC transit and conceivably originated from a B‐cell subset corresponding to post‐GC B cells. Conversely, another fraction of these lymphomas were devoid of somatic IgVH mutations and appeared to originate from naive B cells that have undergone preterminal differentiation independent of GC transit.

Frequent aberrant promoter hypermethylation of O6-methylguanine-DNA methyltransferase and death-associated protein kinase genes in immunodeficiency-related lymphomas.

Summary.  Aberrant promoter hypermethylation is a mechanism of tumour suppressor gene inactivation. We explored aberrant promoter hypermethylation of multiple genes in 88 human immunodeficiency virus (HIV)‐non Hodgkin lymphomas (NHL), 25 post‐transplant lymphoproliferative disorders (PTLD) and five common variable immunodeficiency (CVI)‐related NHL. Twenty‐six of 79 (32·9%) HIV‐NHL, eight of 14 (57·1%) PTLD and two of five (40·0%) CVI–NHL showed aberrant hypermethylation of O6‐methylguanine‐DNA methyltransferase (MGMT). Aberrant hypermethylation of death‐associated protein‐kinase (DAP‐K) occurred in 70 of 84 (83·3%) HIV–NHL, 19 of 25 (72·0%) PTLD and three of five (60·0%) CVI–NHL. These data implicate MGMT and DAP‐K hypermethylation in lymphomagenesis of immunodeficient hosts. In particular, promoter hypermethylation of DAP‐K represents the most frequent molecular alteration yet identified in immunodeficiency‐related lymphomas.

Evidence of biased immunoglobulin variable gene usage in highly stable B-cell chronic lymphocytic leukemia

Recognition of biased immunoglobulin variable (IgV) gene usage in B-cell chronic lymphocytic leukemia (B-CLL) may yield insight into leukemogenesis and may help to refine prognostic categories. We explored Ig variable heavy (VH) and light (VL) chain gene usage in highly stable and indolent B-CLL (n=25) who never required treatment over 10 or more years. We observed an unexpectedly high usage of mutated VH3-72 (6/25; 24.0%), a gene that was otherwise rare in B-CLL (7/805; 0.87%; P<0.01), including mutated cases (6/432; 1.39%; P<0.01) and was exceptional among indolent (1/230, 0.435%; P<0.01), and aggressive B-cell lymphomas (0/105; P<0.01). Three of six VH3-72 B-CLL cases utilized the same VL Vκ4-1 gene. Two VH3-72 B-CLL cases had highly homologous VH complementarity determining regions 3 (CDR3s), encoding Cys-XXXX-Cys domains, and utilized Vκ4-1 genes with homologous IgVL CDR3s. An identical threonine to isoleucine change at codon 84 of VH3-72 framework region 3 (FR3) recurred in four cases of highly stable VH3-72 B-CLL. This mutation is expected to cause a conformational change of FR3 proximal to CDR3 that might critically affect high-affinity antigen binding. B-cell receptors encoded by VH3-72 may identify a specific B-CLL group and be implicated in leukemogenesis through an antigen-driven expansion of B cells.

Aberrant somatic hypermutation in post‐transplant lymphoproliferative disorders

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Analysis of aberrant somatic hypermutation (SHM) in non-Hodgkin's lymphomas of patients with chronic HCV infection

Hepatitis C virus (HCV) and aberrant somatic hypermutation (SHM) have each been suggested to contribute to the development of B‐cell non‐Hodgkins lymphoma (NHL). The incidence of PIM‐1, PAX‐5, RhoH/TTF, and c‐MYC mutations in tumour biopsy specimens from 32 HCV‐infected B‐cell NHL patients was analysed to determine whether the extent of aberrant SHM among these patients differed from that previously reported for HCV‐negative B‐cell NHL patients. Mutation of PIM‐1, PAX‐5, RhoH/TTF, and c‐MYC was detected in 4 (13%), 5 (16%), 4 (13%), and 4 (13%) of 32 samples, respectively. In HCV‐positive B‐cell NHL patients, the frequency of aberrant SHM was lower than that already found in HCV‐negative B‐cell NHL patients. This indicates that, unlike B‐cell lymphomas from HCV‐negative patients, aberrant SHM may not contribute significantly to malignant transformation in HCV‐associated B‐cell lymphomas. Copyright

Aberrant somatic hypermutation in primary mediastinal large B-cell lymphoma.

Primary mediastinal large B-cell lymphoma (PMLBCL) is recognized as a subtype of diffuse large B-cell lymphoma (DLBCL) arising in the mediastinum. However, the clinical, morphological and molecular peculiarities of PMLBCL have been taken to suggest that the disease may represent a distinct clinico-pathologic entity. Clinically, PMLBCL patients display younger median age (35 vs 60 years) and a higher female:male ratio when compared to DLBCL. Also, PMLBCL rarely has extrathoracic disease at diagnosis, and at relapse preferentially involves extranodal sites. Pathologically, PMLBCL is typically associated with various degrees of sclerosis and consists of a diffuse proliferation of large cells expressing B-lineage markers but lacking surface immunoglobulins. At the molecular level, PMLBCL exhibits several genetic abnormalities, including gains of 9p and X that are otherwise absent in DLBCL, and lack genetic lesions, such as BCL-2 and BCL-6 rearrangements that are typically associated with DLBCL. An aberrant activity of the somatic hypermutation (SHM) mechanism, targeting the 50 sequences of PIM-1, PAX-5, RhoH/ TTF and c-MYC proto-oncogenes, has been advocated as a molecular feature distinctive of DLBCL. Aberrant SHM affects 450% of DLBCL of immunocompetent hosts, while it is rare or absent among other B-cell malignancies. The involvement of aberrant SHM in PMLBCL has been suggested by a recent report based on a limited number of cases. These observations prompted our comprehensive analysis aimed at exploring the involvement of aberrant SHM of PIM-1, PAX-5, RhoH/TTF and c-MYC in a sizeable panel of PMLBCL and at comparing the mutational spectrum with that of systemic DLBCL. This study was based on 19 primary samples of PMLBCL. The clinical characteristics of this series were consistent with PMLBCL diagnosis (median age of 37 years; male:female ratio of 0.8; Ann Arbor stage I–II). Diagnosis was pathologically confirmed by three expert hematopathologists (MP, AC and SAP) according to the World Health Organization classification of hematopoietic tumours. Malignant cells expressed CD20 in all cases, CD30 in 16 cases and CD23 in 13 cases, but lacked CD15. For comparison, 19 systemic DLBCL, matched for stage, sex and age, were also analysed. Mutations of PIM-1, PAX-5, RhoH/TTF and c-MYC genes were detected by DNA direct sequencing. The presence of intraclonal heterogeneity, indicative of ongoing SHM, was assessed by sequencing cloned PCR products generated using the proof-reading Pfu Turbo polymerase. The prevalence of aberrant SHM in PMLBCL and DLBCL was compared by the w test. Differences in the mutation frequency between PMLBCL and DLBCL were analysed by the t-test. Mutation frequencies were normalized based on the nucleotide composition of the sequences analysed. The normalized mutation frequency of nucleotides occurring in the context of an RGYW/WRCY motif was compared to the expected mutation frequency by the goodness-of fit w test. Mutation analysis of PAX-5, Rho/TTF, PIM-1 and c-MYC is summarized in Table 1. Overall, the prevalence of aberrant SHM did not significantly differ between PMLBCL and DLBCL. Mutations targeting at least one of the four proto-oncogenes were found in 14/19 (73.6%) PMLBCL and 13/19 (68.4%) DLBCL, while mutations targeting more than one gene were found in 7/19 (36.8%) PMLBCL and 9/19 (47.3%) DLBCL. Each of the four genes analysed was altered in a significant fraction of PMLBCL and DLBCL, since PAX-5 was mutated in 9/19 (47.3%) PMLBCL and 7/19 (36.8%) DLBCL, RhoH/TTF in 6/19 (31.5%) PMLBCL and 8/19 (42.1%) DLBCL, PIM-1 in 3/19 (15.7%) PMLBCL and 7/19 (36.8%) DLBCL, and c-MYC in 6/19 (31.5%) PMLBCL and 5/19 (26.3%) DLBCL. The detailed characterization of PAX-5, RhoH/TTF, PIM-1 and c-MYC mutations is reported in Tables 1 and 2. Overall, the molecular profile of mutations was similar between PMLBCL and DLBCL. A total of 74 mutational events were detected in PMLBCL. The overwhelming majority of the mutations included single base-pair substitutions (n1⁄4 66), whereas deletions of a short DNA stretch were observed in eight instances. Of the 66 single base-pair substitutions observed, 41 were transitions and 25 were transversions, with a transition/transversion ratio of 1.64 (expected 0.5; P1⁄4 0.001). When considering all the nucleotide substitutions observed, analysis of the nucleotide exchange pattern showed that GþC base pairs were targeted 3.33-fold more frequently than Aþ T (expected 1.28; P1⁄4 0.009). In all, 19 of 66 single base-pair substitutions detected in PMLBCL felt within RGYW/WRCY motifs. Considering all the genes together, the frequency of mutations targeting RGYW/ WRCY motifs was significantly higher than the frequency of mutations occurring outside RGYW/WRCY motifs (2.3 vs 1.3%; P1⁄4 0.03). As mutations introduced by physiological SHM preferentially affect specific dinucleotides, we also analysed the distribution of mutations within dinucleotide motifs. Considering together all the four proto-oncogenes, the mutation frequency was higher than expected in the following motifs: AA (4.3 vs 1.3%; Po0.0001), AG (3.0 vs 1.3%; P1⁄4 0.028), GC (5.1 vs 1.1%; Po0.0001), GG (4.2 vs 1.1%; Po0.0001) and TA (4.6 vs 1.3%; P1⁄4 0.003). Among DLBCL, a total of 87 mutational events were detected. Mutations were preferentially represented by single base-pair substitutions (n1⁄4 81), whereas only four deletions and two insertions of a short DNA stretch were observed. Of the 81 single base-pair substitutions, 42 were transitions and 39 were transversions, with a transition/transversion ratio of 1.07 (expected 0.5; P1⁄4 0.02). Analysis of the nucleotide exchange pattern showed that GþC base pairs were targeted 1.89-fold more frequently compared to Aþ T (expected 1.28; P1⁄4 ns). In all, 41 out of 81 single base-pair substitutions felt within RGYW/WRCY motifs. The frequency of mutations targeting RGYW/WRCY motifs was significantly higher than the frequency of mutations occurring outside RGYW/WRCY motifs (5.0 vs 1.7%; Po0.0001). Similarly to PMLBCL, also in DLBCL the mutation frequency was higher than expected in specific dinucleotide motifs: AA (6.4 vs 1.5%; Po0.0001), AC (5.4 vs 1.4%; Po0.0001), AG (4.5 vs 1.6%; Po0.0001), AT (4.2 vs 1.7%; P1⁄4 0.033), CT (4.4 vs 1.6%; Received 6 April 2005; accepted 6 September 2005; published online 6 October 2005 Correspondence: Professor G Gaidano, Division of Hematology, Department of Medical Sciences & IRCAD, Amedeo Avogadro University of Eastern Piedmont, Via Solaroli 17, 28100 Novara, Italy; Fax þ 39 0321 620 421; E-mail: gaidano@med.unipmn.it Correspondence

Incidence of novel N-glycosylation sites in the B-cell receptor of lymphomas associated with immunodeficiency

Novel N‐glycosylation sites are introduced by somatic mutation into the V genes of the majority of follicular lymphomas. Sites are positively selected and rare in normal memory B cells, indicating a potential role in tumour survival in the germinal centre (GC). The incidence of c. 40% in diffuse large B‐cell lymphomas (DLBCL) parallels the known heterogenity of the disease. Immunodeficiency‐related non‐Hodgkins lymphomas (NHL) include post‐transplant lymphoproliferative disorders (PTLD) and acquired immunodeficiency syndrome‐related NHL (AIDS‐NHL). Most PTLD derive from B cells that carry mutated VH genes and that have completed the GC reaction. All AIDS‐NHL carry mutated VH genes and variable features of GC or post‐GC cells. To determine if N‐glycosylation is a feature of immunodeficiency‐related lymphomas, we analysed the VH genes of 19 PTLD and 36 AIDS‐NHL. Novel sites were rare in PTLD (4/19), similar to memory B cells (P = 0·15). AIDS‐NHL, including DLBCL and Burkitts lymphomas (BL), showed heterogeneity with 16 of 36 (44%) having novel sites. The findings indicate no selection of N‐glycosylation sites in PTLD, consistent with post‐GC features. The variable incidence of N‐glycosylation sites in AIDS‐NHL mirrors that in DLBCL and sporadic BL of immunocompetent hosts, supporting the known heterogeneity of these disorders, and possibly pointing to distinct routes of tumour development.