Blastic variant of mantle cell lymphoma shows a heterogenous pattern of somatic mutations of the rearranged immunoglobulin heavy chain variable genes

Mantle cell lymphoma is a distinct clinicopathological entity associated with t(11;14) and cyclin D1 overexpression. The majority of cases show uniform morphological and phenotypic features characterized by a monotonous proliferation of small-to-medium-sized irregular B cells that express CD5 and bright surface immunoglobulin IgM and IgD. By sequence analysis of the rearranged immunoglobulin heavy chain variable genes (VH), it has been shown that these lymphoma cells carry little if no somatic mutations, as described for the fetal CD5⁺ cells or B1 cells. Besides mantle cell lymphoma with classic histological features, a morphological variant of mantle cell lymphoma with blastic features and a more aggressive clinical course has been described. To investigate whether this variant is closely related, by the cell of origin, to typical cases, we analysed the presence and the pattern of somatic mutations of the VH genes in a series of nine cases diagnosed as such. Our cases of blastic mantle cell lymphomas rearrange most frequently VH4 and VH3 family genes. In three cases there was a complete homology to published germline genes, and a near complete homology was documented in another three. In contrast, the remaining three cases showed somatic mutations in their rearranged VH genes. Mutation analysis revealed evidence for antigen selection in one of these three cases. Taken together, these data are similar to those of normal adult-type B1 cells and those described for chronic lymphocytic leukaemia (CLL) but slightly different to those reported for classic mantle cell lymphoma. It is likely that blastic mantle cell lymphoma as well as CLL originates from adult-type B1 cells. More cases will need to be studied to determine whether classic mantle cell lymphoma is different from the blastic subtype and if it arises from fetal-type B1 cells.     

Ongoing immunoglobulin gene mutations in mantle cell lymphomas

Mantle cell lymphomas (MCL) frequently show a vaguely follicular growth pattern. This phenomenon is thought to result from the colonization of reactive B-cell follicles by tumour cells. In view of the unique property of the germinal centre environment, antigen stimulation may play a role in the expansion of the tumour. To assess this, we have examined ongoing Ig mutations, which are genetic markers of B cells in persistent response to antigen stimulation, in five MCLs including two cases derived from the gastrointestinal tract known as lymphomatous polyposis (LP). We have specifically analysed Ig ongoing mutations in tumour cells from multiple lesions in one case and in tumour cells microdissected from colonized follicles in two cases. The consensus Ig V_H sequences in four MCLs were identical, or almost identical (three cases 100%, one case 99% homology) to the published germlines, which in each case were those frequently employed by autoantibodies. The consensus Ig V_H sequence in the remaining case displayed 95.5% homology to the closest published germline. This may represent derivation from an unknown V_H germline or a rare instance of somatic mutations. Extensive sequencing of the rearranged Ig genes revealed ongoing mutations within the tumour clone in two cases: one was a LP with multiple lesions of the gastrointestinal tract and the other was a nodal MCL in which tumour cells from colonized follicles were analysed. Our results indicate that MCLs are derived from pre-germinal centre B cells, possibly autoreactive B-cell clones. The ongoing mutations identified suggest a possible involvement of antigen stimulation in the clonal expansion of a proportion of MCLs.     

Hodgkin's disease with a B-cell phenotype often shows a VDJ rearrangement and somatic mutations in the VH genes

The nature of Hodgkin and Reed-Sternberg (HRS) cells remains in question. Immunophenotypic studies favor a relation to the lymphoid lineage with the existence of B- and T-cell types. However, studies on the detection of antigen (Ag) receptor gene rearrangements provided inconsistent results. They concur in that rearranged Ig and T-cell receptor (TCR) genes are not demonstrable in most Hodgkin's disease (HD) cases. To clarify whether this is because of the insensitivity of the method of detection or a real absence of clonal Ig heavy chain (IgH) rearrangements, a polymerase chain reaction (PCR) method with high sensitivity was applied, allowing the detection of less than 50 cells with clonally rearranged IgH genes in a mixture of 100,000 germline or individually rearranged cells. In 67 cases of HD, most of those (67%) with B-Ag+ HRS cells express clonal VDJ rearrangements of the IgH gene. No cases with T-cell Ag+ HRS cells harbored detectable clonal VDJ rearrangements. Of 10 sequenced rearranged IgH genes, the VH segment of six contained considerable somatic mutations. These results suggest that the demonstrated VDJ rearrangements stem from the HRS cells themselves and that the HRS cells of cases with rearranged IgH genes are B-cell related and correspond in their differentiation stage either to naive pregerminal center B cells or (more commonly) to germinal center/postgerminal center-derived memory B cells.     

Hodgkin and Reed-Sternberg-like cells in B-cell chronic lymphocytic leukemia represent the outgrowth of single germinal-center B-cell-derived clones: potential precursors of Hodgkin and Reed-Sternberg cells in Hodgkin's disease

In rare cases of B-cell chronic lymphocytic leukemia (B-CLL), large cells morphologically similar to or indistinguishable from Hodgkin/Reed-Sternberg (HRS) cells of Hodgkin's disease (HD) can be found in a background of otherwise typical B-CLL. To test these HRS-like cells for a potential clonal relationship to the B-CLL cells, single cells were micromanipulated from immunostained tissue sections, and rearranged immunoglobulin genes were amplified from HRS-like cells and B-CLL cells and sequenced. The same variable (V) gene rearrangements with shared and distinct somatic mutations were found in HRS-like and B-CLL cells from 1 patient, which indicates derivation of these cells from 2 distinct members of a germinal-center B-cell clone. Separate clonal V gene rearrangements were amplified from HRS-like and B-CLL cells from 2 other patients, showing concomitant presence of 2 distinct expanded B-cell clones. Epstein-Barr virus (EBV) was detected in the HRS-like cells of these 2 latter cases, indicating clonal expansion of an EBV-harboring B cell in the setting of B-CLL. There is evidence that HRS-like cells in B-CLL, like HRS cells in HD, derive from germinal-center B cells. In all cases, somatic mutations have been detected in the rearranged V genes of the HRS-like cells, and in 1 of the EBV-positive HRS-like cell clones, somatic mutations rendered an originally functional V gene rearrangement nonfunctional. We speculate that the HRS-like cells in B-CLL represent potential precursors for HRS cells causing HD.     

Kaposi sarcoma-associated herpesvirus infects monotypic (IgM lambda) but polyclonal naive B cells in Castleman disease and associated lymphoproliferative disorders

In a previous study, it was shown that the Kaposi sarcoma-associated herpesvirus (KSHV) was specifically associated with monotypic (IgMlambda) plasmablasts in multicentric Castleman disease (MCD). The plasmablasts occur as isolated cells in the mantle zone of B-cell follicles but may form microlymphoma or frank plasmablastic lymphoma. To determine the clonality and cellular origin of the monotypic plasmablasts, the rearranged Ig genes in 13 patients with KSHV-related MCD, including 8 cases with microlymphomas and 2 with frank lymphomas, were studied. To investigate the role of the interleukin 6 (IL-6) receptor signaling in the pathogenesis of MCD and associated lymphoproliferative disorders, viral IL-6 and human IL-6 receptor expression was examined. KSHV-positive plasmablasts were polyclonal in MCD-involved lymphoid tissues in all cases and microlymphomas in 6 of 8 cases. Monoclonal KSHV-positive plasmablasts were seen in microlymphomas of 2 cases and in both frank lymphomas. Despite their mature phenotype, KSHV-positive plasmablasts did not harbor somatic mutations in the rearranged Ig genes, indicating origination from naive B cells. Viral IL-6 was expressed in 10% to 15% of KSHV-positive plasmablasts, whereas the human IL-6 receptor was expressed in most KSHV-positive cells. Thus, KSHV infects monotypic but polyclonal naive B cells and is associated with a range of lymphoproliferative disorders from polyclonal isolated plasmablasts and microlymphomas to monoclonal microlymphoma and frank plasmablastic lymphomas in MCD patients. Activation of the IL-6 receptor signaling pathway may play a role in differentiation of KSHV-infected naive B cells into plasmablasts and development of lymphoproliferative lesions. (Blood. 2001;97:2130-2136)     

Frequent occurrence of deletions and duplications during somatic hypermutation: Implications for oncogene translocations and heavy chain disease

Human naive and germinal center (GC) B cells were sorted by flow cytometry and rearranged V_H region genes were amplified and sequenced from single cells. Whereas no deletions or insertions were found in naive B cells, ≈4% of in-frame and >40% of out-of-frame rearrangements of GC B cells harbored deletions and/or insertions of variable length. The pattern of deletions/insertions and their restriction to mutated V genes strongly suggests that they result from somatic hypermutation. Deletions and insertions account for ≈6% of somatic mutations introduced into rearranged V_H region genes of GC B cells. These deletions/insertions seem to be the main cause for the generation of heavy chain disease proteins. Furthermore, it appears that several types of oncogene translocations (like c-myc translocations in Burkitt’s lymphoma) occur as a byproduct of somatic hypermutation within the GC—and not during V(D)J recombination in the bone marrow as previously thought.     

Epstein-Barr virus-infected B cells expanding in germinal centers of infectious mononucleosis patients do not participate in the germinal center reaction

To assess the impact of the germinal center (GC) reaction on viral spread in Epstein-Barr virus (EBV) infection, we isolated EBV(+) GC B cells from the tonsils of two infectious mononucleosis patients, sequenced their rearranged V genes, and determined expression of the EBV latency genes EBV nuclear antigen 2 and latent membrane protein 1. Most EBV(+) GC B cells belonged to clones of cells harboring somatically mutated V gene rearrangements. Ongoing somatic hypermutation, the hallmark of the GC reaction, was seen only in uninfected GC B cell clones, not in EBV(+) B cell clones. Thus, in infectious mononucleosis, GC and/or memory B cells are directly infected by EBV and expand without somatic hypermutation, whereas the GC passage of EBV-infected naive B cells does not contribute detectably to the generation of infected memory B cells, the main reservoir of EBV during persistence. Most, if not all, EBV-infected cells in GCs exhibited an unusual EBV gene expression pattern in that they were positive for EBV nuclear antigen 2 but negative for latent membrane protein 1. Although the three main types of EBV-associated B cell lymphomas (Burkitt's, Hodgkin's, and posttransplant lymphomas) presumably are derived from GC B cells, EBV(+) GC B cells resembling these EBV(+) GC B cell lymphomas in terms of EBV gene expression and somatic hypermutation pattern could not be identified.     

Rescue of "crippled" germinal center B cells from apoptosis by Epstein-Barr virus

Epstein-Barr virus (EBV) is associated with B-cell lymphomas such as Hodgkin lymphoma, Burkitt lymphoma, and post-transplantation lymphoma, which originate from clonal germinal center (GC) B cells. During the process of somatic hypermutation, GC B cells can acquire deleterious or nonsense mutations in the heavy and light immunoglobulin genes. Such mutations abrogate the cell surface expression of the B-cell receptor (BCR), which results in the elimination of these nonfunctional B cells by immediate apoptosis. EBV encodes several latent genes, among them latent membrane protein 1 (LMP1) and LMP2A, which are regularly expressed in EBV-positive Hodgkin lymphoma and posttransplantation lymphomas. Since LMP1 and LMP2A mimic the function of 2 key receptors on B cells, CD40 and BCR, respectively, we wanted to learn whether EBV infection can rescue proapoptotic GC B cells with crippling mutations in the heavy chain immunoglobulin locus from apoptosis. We show here that BCR-negative GC B cells readily enter the cell cycle upon infection with EBV in vitro and yield clonal lymphoblastoid cell lines that are incapable of expressing a functional BCR because the rearranged and formerly functional heavy chain immunoglobulin alleles carry deleterious mutations. Our findings imply an important role for EBV in the process of lymphomagenesis in certain cases of Hodgkin lymphoma and posttransplantation lymphomas.     

Monocytoid B cells are distinct from splenic marginal zone cells and commonly derive from unmutated naive B cells and less frequently from postgerminal center B cells by polyclonal transformation.

Monocytoid B cells represent a morphologically conspicuous B-cell population that constantly occurs in Toxoplasma gondii-induced Piringer's lymphadenopathy. Although widely believed to be closely related to splenic marginal zone B cells, neither this relationship, nor the B-cell differentiation stage of monocytoid B cells, nor their cellular precursors have been established. We have therefore examined monocytoid B cells for their expression of B-cell differentiation markers and the Ig isotypes at the RNA and protein level as well as for rearranged Ig heavy chain (H) genes and somatic mutations within the variable (V) region. The results obtained were compared with the corresponding features of other B-cell populations. The monocytoid B cells displayed immunophenotypical differences to all other B-cell populations. IgM and IgD expression was absent from most monocytoid B cells at the RNA and protein levels. Unrelated (polyclonal) Ig rearrangements were found in 85 of the 95 cells studied. Seventy-four percent of the rearranged VH genes were devoid of somatic mutations, whereas the remaining 26% carried a low number of somatic mutations. The majority of these showed no significant signs of antigen selection. This finding in conjunction with the predominantly unrelated Ig gene rearrangements indicates that most monocytoid B cells arise not by clonal proliferation but by transformation of polyclonal B cells. The B cells undergoing a monocytoid B-cell transformation are in the majority (74%) naive B cells, and only a minority are (26%) non-antigen-selected postgerminal center B cells. Thus, our data show that monocytoid B cells represent a distinct B-cell subpopulation.     

Analysis of VH gene rearrangements from synovial B cells of patients with rheumatoid arthritis reveals infiltration of the synovial membrane by memory B cells

The VH gene (Variable gene segments of the heavy chain locus) repertoire can be investigated by DNA analysis of rearranged immunoglobulin VH genes, which also allows for an indirect estimation of antibody selection by analysis of somatic mutations. Using a polymerase chain reaction (PCR) it is also possible to analyse these genes in small numbers of cells or even single cells. This approach was chosen to investigate germinal centre like lymphocyte follicles in the synovial membranes of two patients with rheumatoid arthritis (RA) in order to analyse the local humoral immune response in RA. Individual B-cell aggregates of synovial membrane of two patients with RA were isolated by micromanipulation from microscopic slides. VH-DH-JH (variable, diversity, and joining segments of the heavy chain locus) rearrangements in all possible VH-JH combinations were amplified from these B cell foci, cloned and subjected to sequence analysis. Sequence analysis revealed that most of the rearranged VH genes were somatically mutated with at least 1% (range 1.3 – 14.9%) somatic mutations and therefore were derived from antigen-selected memory B cells. Intraclonal diversity in one-third of the clones indicated the generation of memory B cells in the synovial membrane and characterized the synovial membrane as lymphatic tissue where secondary immune responses to an as yet unknown antigen take place. Received: 22 April 1997 / Accepted: 25 August 1997     

VH gene mutation status and cellular drug resistance in chronic lymphocytic leukaemia

OBJECTIVE: B-cell chronic lymphocytic leukaemia (B-CLL) can be divided into two clinical entities based on the immunoglobulin variable heavy chain (VH) gene mutation status, as cases with unmutated VH genes display a more aggressive disease with shorter survival time than mutated cases. The aim of this study was to investigate whether differences in cellular drug resistance could give an explanation for these divergent clinical courses. METHODS: The VH gene mutation status was analysed in patients with previously untreated B-CLL using VH gene family-specific PCR amplification and nucleotide sequencing. In vitro sensitivity to cytarabine, fludarabine, cladribine, doxorubicin, idarubicin, vincristine, cyclophosphamide, melphalan and prednisolone was assessed using the non-clonogenic in vitro assay, fluorometric microculture cytotoxicity assay. RESULTS: The VH genes and in vitro drug resistance were successfully analysed in 46 cases, revealing that 25 (54%) cases showed unmutated and 21 (46%) cases mutated VH genes. Interestingly, the unmutated group generally tended to be more chemosensitive than the mutated group with significant differences for cytarabine and prednisolone (P < or = 0.01). CONCLUSION: The propensity of inferior drug response in mutated B-CLL may reflect a more differentiated disease than in unmutated B-CLL. We conclude that the difference in prognosis between B-CLL cases with unmutated and mutated VH genes could not be explained by difference in cellular drug resistance.     

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 heterogeneity of the disease. Immunodeficiency-related non-Hodgkin's 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 Burkitt's 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.     

Molecular analysis of single B cells from T-cell-rich B-cell lymphoma shows the derivation of the tumor cells from mutating germinal center B cells and exemplifies means by which immunoglobulin genes are modified in germinal center B cells

T-cell-rich B-cell lymphoma (TCRBCL) belongs to the group of diffuse large cell lymphomas (DLL). It is characterized by a small number of tumor B cells among a major population of nonmalignant polyclonal T cells. To identify the developmental stage of the tumor progenitor cells, we micromanipulated the putative neoplastic large CD20(+) cells from TCRBCLs and amplified and sequenced immunoglobulin (Ig) V gene rearrangements from individual cells. In six cases, clonal Ig heavy, as well as light chain, gene rearrangements were amplified from the isolated B cells. All six cases harbored somatically mutated V gene rearrangements with an average mutation frequency of 15.5% for heavy (VH) and 5.9% for light (VL) chains and intraclonal diversity based on somatic mutation. These findings identify germinal center (GC) B cells as the precursors of the transformed B cells in TCRBCL. The study also exemplifies various means how Ig gene rearrangements can be modified by GC B cells or their malignant counterparts in TCRBCL: In one case, the tumor precursor may have switched from kappa to lambda light chain expression after acquiring a crippling mutation within the initially functional kappa light chain gene. In another case, the tumor cells harbor two in-frame VH gene rearrangements, one of which was rendered nonfunctional by somatic mutation. Either the tumor cell precursor entered the GC with two potentially functional in-frame rearrangements or the second VHDHJH rearrangement occurred in the GC after the initial in-frame rearrangement was inactivated by somatic mutation. Finally, in each of the six cases, at least one cell contained two (or more) copies of a clonal Ig gene rearrangement with sequence variations between these copies. The presence of sequence variants for V region genes within single B cells has so far not been observed in any other normal or transformed B lymphocyte. Fluorescence in situ hybridization (FISH) points to a generalized polyploidy of the tumor cells.     

Analysis of expressed immunoglobulin heavy chain genes in familial B-CLL

In this study, we wished to determine whether familial chronic lymphocytic leukemia of B-cell phenotype (CLL) shares with sporadic B-CLL the same immunoglobulin (Ig) heavy chain variable region (VH) gene usage and occurrence of somatic mutation, to gain insight into the pathogenetic relatedness of these epidemiologically distinct forms of CLL. We therefore analyzed the expressed Ig heavy chain genes in 23 cases (11 families) of familial CLL, and compared these results with data previously reported for sporadic CLL. In addition, we assessed the relationship of the occurrence of somatic mutation to several clinical and phenotypic features. The distribution of V genes among these cases was similar to that observed in sporadic CLL: VH3 > VH1 > VH4. Thirteen of the 23 cases (57%) showed germ line VH gene sequences, whereas somatic mutations were detected in 10 cases (43%). The average mutation frequency of these latter 10 cases of was 6.7% (ranging from 1.7% to 8.8%), and evidence of antigen selection was noted in 6. Intraclonal variation, followed by clonal evolution and the appearance of a second clone over a 20-year period was observed in 1 case, suggesting that mutations can continue to accumulate after neoplastic transformation. The presence of somatic mutations correlated with age at presentation, low white blood cell (WBC) count, and low fluorescence intensity of surface CD5, and the potential significance of these relationships is discussed. Our data indicate that familial and sporadic B-CLL display a similar pattern of immunoglobulin gene usage and frequency of somatic mutation, and are consistent with a common ontogeny and immunogenetic origin for these 2 epidemiologically distinct forms of CLL. (Blood. 2000;95:1413-1419)     

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.     

Analysis of VH genes used by neoplastic B cells in endemic Burkitt's lymphoma shows somatic hypermutation and intraclonal heterogeneity

Tumor cell lines from six typical cases of endemic Epstein-Barr virus (EBV) genome-positive Burkitt's lymphoma (BL) have been investigated for usage and mutational pattern of Ig VH genes. The neoplastic cells all had a t(8;14) (q24;q32) translocation involving the c-myc protooncogene. The VH genes were derived from VH1, VH3 and VH4, and both the IgM-positive (four cases) and IgG-positive (two cases) were extensively mutated from germline sequence. In two cases, early and late passage tumor cells were available, and the VH nucleotide sequences were identical, indicating that mutations had not accumulated in vitro. In a further case, there was evidence of sequence heterogeneity, which appeared to have been generated in vivo, indicating that the tumor cell VH gene was able to undergo posttranslocation somatic hypermutation. Analysis of the relatively nonpolymorphic VH4 genes for the pattern of replacement or silent mutations did not show a role for antigen selection in the expressed sequences.     

Somatic mutation of bcl-6 genes can occur in the absence of V(H) mutations in chronic lymphocytic leukemia

Somatic mutation in immunoglobulin variable (V) region genes occurs largely in the germinal center and, after neoplastic transformation, imprints V genes of B-cell tumors with the mutational history of the cell of origin. Recently, it has been found that chronic lymphocytic leukemia (CLL) consists of 2 subsets, each with a different clinical course, one with unmutated V(H) genes consistent with a naive B cell, and the other with mutated V(H) genes consistent with transit through the germinal center. However, somatic mutation also occurs at another distinct locus, the 5' noncoding region of the bcl-6 gene, in both B-cell tumors and in normal germinal center B cells. To probe the suggestive link between the occurrence of mutations in V(H) and bcl-6 genes, we analyzed the nature of somatic mutation at these distinct loci in the 2 CLL subsets. Unexpectedly, we found no such link in the CLLs defined by unmutated V(H) genes, with 4 of 10 cases clearly showing mutations in bcl-6. In those CLLs defined by somatically mutated V(H) genes, 4 of 9 cases predictively showed bcl-6 mutations. The frequency of bcl-6 mutations was comparable in both subsets, with mutations being biallelic, and in 3 of 8 cases indicative of clonal origins. Surprisingly, intraclonal variation, which is not a feature of V(H) genes in CLL, was found in 6 of 8 cases in both subsets. These data indicate that somatic mutation of the V(H) and bcl-6 loci may not necessarily occur in tandem in CLL, suggesting diverse pathways operating on the 2 genes.     

Oligonucleotide microarrays demonstrate the highest frequency of ATM mutations in the mantle cell subtype of lymphoma

Mutations have been described in the ataxia telangiectasia mutated (ATM) gene in small numbers of cases of lymphoid neoplasia. However, surveys of the ATM mutation status in lymphoma have been limited due to the large size (62 exons) and complex mutational spectrum of this gene. We have used microarray-based assays with 250,000 oligonucleotides to screen lymphomas from 120 patients for all possible ATM coding and splice junction mutations. The subtypes included were diffuse large B cell, mantle cell, immunoblastic large B cell, follicular, posttransplant lymphoproliferative disorder, and peripheral T cell lymphoma. We found the highest percentage of ATM mutations within the mantle cell (MCL) subtype (43%, 12 of 28 cases), followed by a lower level (10% of cases) in the other subtypes. A frame-shift ATM mutation was found in one peripheral T cell lymphoma patient. In six MCL cases examined, four ATM variants were due to somatic mutation in the tumor cells whereas two others seemed to be germ-line in origin. There was no difference in p53 mutation status in the ATM mutant and wild-type groups of MCL. There was no statistically significant difference in the median overall survival of patients with wild-type vs. mutated ATM in MCL. Additional mutational and functional analyses are needed to determine whether ATM mutations contribute to the development and progression of MCL or are just the consequence of genomic instability in MCL.