Masao Seto

Identification and Characterization of a Novel Gene, C13orf25, as a Target for 13q31-q32 Amplification in Malignant Lymphoma

The amplification at 13q31-q32 has been reported in not only hematopoietic malignancies but also in other solid tumors. We identified previously frequent amplification of chromosomal band 13q31-q32 in 70 cases of diffuse large B-cell lymphoma patients by conventional comparative genomic hybridization analysis. In an attempt to identify a candidate gene within this region, we used array comparative genomic hybridization and fluorescent in situ hybridization to map the 13q31-q32 amplicon. We then screened the 65 expressed sequence tags and Glypican 5 (GPC5) by reverse transcription-PCR and Northern blotting. As a result, we identified a novel gene, designated Chromosome 13 open reading frame 25 (C13orf25), which was overexpressed in B-cell lymphoma cell lines and diffuse large B-cell lymphoma patients with 13q31-q32 amplifications. However, GPC5, which has been reported to be a target gene for 13q31-q32 amplification, was truncated in one cell line, Rec1, possessing the amplification, and its expression in various cell lines with amplification at 13q31-q32 was not significantly different from that in other cell lines without amplification, suggesting that GPC5 is not likely to be the candidate gene. Additional analysis identified two major transcripts in the C13orf25 gene. The two transcripts A and B predicted open reading frames of 32 and 70-amino acid polypeptides, respectively. The former has been reported as bA121J7.2, which is conserved among species. Transcript-B also contained seven mature microRNAs in its untranslated region. These results suggest that the C13orf25 gene is the most likely candidate gene for the 13q31-q32 amplicon found in hematopoietic malignancies.

A novel gene, MALT1 at 18q21, is involved in t(11;18) (q21;q21) found in low-grade B-cell lymphoma of mucosa-associated lymphoid tissue.

The t(11;18) (q21;q21) translocation is a characteristic chromosomal aberration in low-grade B-cell lymphoma of mucosa-associated lymphoid tissue (MALT) type. We previously identified a YAC clone y789F3, which includes the breakpoint at 18q21 in a MALT lymphoma patient. BAC and PAC contigs were constructed on the YAC, and BAC 193f9 was found to encompass the breakpoint region. In the present study, we further narrowed down the breakpoint region at 18q21 in five MALT lymphoma patients by means of FISH and Southern blot analyses using the plasmid contig constructed from BAC 193f9. The breakpoints at 18q21 in three of the five MALT lymphoma patients were found to be clustered approximately within the 20 kb region. By using exon amplification and cDNA library screening, we identified a novel cDNA spanning the breakpoint region that exhibited aberrant mRNA signals in four of the five MALT lymphoma patients. The nucleotide sequence predicted an 813 amino acid protein that shows significant sequence similarity to the CD22β and laminin 5 α3b subunit. We refer to the gene encoding this transcript as MALT1 (Mucosa-Associated Lymphoid Tissue lymphoma translocation gene 1). The alteration of MALT1 by translocation strongly suggests that this gene plays an important role in the pathogenesis of MALT lymphoma.

Genome-wide array-based CGH for mantle cell lymphoma: identification of homozygous deletions of the proapoptotic gene BIM

Mantle cell lymphoma (MCL) is characterized by 11q13 chromosomal translocation and CCND1 overexpression, but additional genomic changes are also important for lymphomagenesis. To identify the genomic aberrations of MCL at higher resolutions, we analysed 29 patient samples and seven cell lines using array-based comparative genomic hybridization (array CGH) consisting of 2348 artificial chromosome clones, which cover the whole genome at a 1.3 mega base resolution. The incidence of identified genomic aberrations was generally higher than that determined with chromosomal CGH. The most frequent imbalances detected by array CGH were gains of chromosomes 3q26 (48%), 7p21 (34%), 6p25 (24%), 8q24 (24%), 10p12 (21%) and 17q23 (17%), and losses of chromosomes 2p11 (83%), 11q22 (59%), 13q21 (55%), 1p21–p22 (52%), 13q34 (52%), 9q22 (45%), 17p13 (45%), 9p21 (41%), 9p24 (41%), 6q23–q24 (38%), 1p36 (31%), 8p23 (34%), 10p14 (31%), 19p13 (28%), 5q21 (21%), 22q12 (21%), 1q42 (17%) and 2q13 (17%). Our analyses also detected several novel recurrent regions of loss located at 1p36, 1q42.2–q43, 2p11.2, 2q13, 17p13.3 and 19p13.2–p13.3, as well as recurrent regions of homozygous loss such as 2p11 (Igκ), 2q13 and 9p21.3–p24.1 (INK4a/ARF). Of the latter, we investigated the 2q13 loss, which led to identification of homozygous deletions of the proapoptotic gene BIM. The high-resolution array CGH technology allowed for the precise identification of genomic aberrations and identification of BIM as a novel candidate tumor suppressor gene in MCL.

Molecular signatures to improve diagnosis in peripheral T-cell lymphoma and prognostication in angioimmunoblastic T-cell lymphoma.

Peripheral T-cell lymphoma (PTCL) is often challenging to diagnose and classify. Gene expression profiling was performed on 144 cases of PTCL and natural killer cell lymphoma and robust molecular classifiers were constructed for angioimmunoblastic T-cell lymphoma (AITL), anaplastic lymphoma kinase-positive (ALK(+)) anaplastic large-cell lymphoma (ALCL), and adult T-cell leukemia/lymphoma. PTCL-unclassifiable was molecularly heterogeneous, but we were able to identify a molecular subgroup with features of cytotoxic T lymphocytes and a poor survival compared with the remaining PTCL-not otherwise specified cases. Many of the pathologic features and substantial components of the molecular signature of AITL are contributed by the follicular dendritic cells, B-cell, and other stromal components. The expression of Th17-associated molecules in ALK(+) ALCL was noted and may represent aberrant activation of Th17-cell differentiation by abnormal cytokine secretion. Adult T-cell leukemia/lymphoma has a homogeneous molecular signature demonstrating high expression of human T-lymphotropic virus type 1-induced genes. These classifiers reflect the biology of the tumor cells as well as their microenvironment. We also constructed a molecular prognosticator for AITL that appears to be largely related to the microenvironmental signature, and the high expression of 2 immunosuppressive signatures are associated with poor outcome. Oncogenic pathways and tumor-host interactions also were identified, and these findings may lead to better therapies and outcome in the future.

Genome-wide microRNA expression profiling in renal cell carcinoma: significant down-regulation of miR-141 and miR-200c.

We investigated expression profiles of microRNA (miRNA) in renal cell carcinoma [clear cell carcinomas (CCC) and chromophobe renal cell carcinomas (ChCC)] and in normal kidneys by using a miRNA microarray platform which covers a total of 470 human miRNAs (Sanger miRBase release 9.1). Unsupervised hierarchical cluster analysis revealed that CCC and ChCC were separable and that no subgroups were identified in CCCs. We found that 43 miRNAs were differentially expressed between CCC and normal kidney, of which 37 were significantly down‐regulated in CCC and the other 6 were up‐regulated. We also found that 57 miRNAs were differentially expressed between ChCC and normal kidney, of which 51 were significantly down‐regulated in ChCC and the other 6 were up‐regulated. Together, these observations indicate that expression of miRNAs tends to be down‐regulated in both CCC and ChCC compared with normal kidney. We observed that miR‐141 and miR‐200c were the most significantly down‐regulated miRNAs in CCCs. Indeed, in all cases of CCC analysed, both miR‐141 and miR‐200c were down‐regulated in comparison with normal kidney. Microarray data and quantitative RT–PCR showed that these two miRNAs were expressed concordantly. TargetScan algorithm revealed that ZFHX1B mRNA is a hypothetical target of both miR‐141 and ‐200c. We established by quantitative RT–PCR that, in CCCs in which miR‐141 and miR‐200c were down‐regulated, ZFHX1B, a transcriptional repressor for CDH1/E‐cadherin, tended to be up‐regulated. Furthermore, we found that overexpression of miR‐141 and miR‐200c caused down‐regulation of ZFHX1B and up‐regulation of E‐cadherin in two renal carcinoma cell lines, ACHN and 786‐O. On the basis of these findings, we suggest that down‐regulation of miR‐141 and miR‐200c in CCCs might be involved in suppression of CDH1/E‐cadherin transcription via up‐regulation of ZFHX1B. Copyright

TNFAIP3/A20 functions as a novel tumor suppressor gene in several subtypes of non-Hodgkin lymphomas

The constitutive activation of nuclear factor-kappaB (NF-kappaB) has been implicated in tumorigenesis of lymphoid malignancies. We have previously shown that chromosome 6q was frequently deleted in ocular marginal zone B-cell lymphoma and identified TNFAIP3/A20, a negative regulator of NF-kappaB pathways, as the primary target for 6q deletion. In the study reported here, we extended the analysis to other subsets of non-Hodgkin lymphomas and found that A20 is frequently deleted in mantle cell lymphoma and diffuse large B-cell lymphoma. Importantly, A20 promoter methylation or gene mutation is also frequently detected in these lymphomas, raising the possibility that inactivation of A20 may be involved in lymphomagenesis. To address this question, we conducted overexpression experiments in lymphoma cell lines with A20 deletion and down-regulated expression of A20 with an siRNA technique in Epstein-Barr virus-infected lymphoblastoid cell lines. These experiments found that overexpression of A20 induced apoptosis and silencing of A20 was associated with resistance to apoptosis and enhanced clonogenicity. The cells with down-regulated A20 exhibited enhanced NF-kappaB activities, which may account for the observed effects. These results indicate that our study provides a novel insight into molecular mechanisms leading to lymphoma and that specific targeting of NF-kappaB pathways may be advantageous for treatment.

Genome‐wide array‐based comparative genomic hybridization of natural killer cell lymphoma/leukemia: Different genomic alteration patterns of aggressive NK‐cell leukemia and extranodal Nk/T‐cell lymphoma, nasal type

Natural killer (NK) cell lymphomas/leukemias are highly aggressive lymphoid malignancies, but little is known about their genomic alterations, and thus there is an urgent need for identification and analysis of NK cell lymphomas/leukemias. Recently, we developed our own array‐based comparative genomic hybridization (array CGH) with an average resolution of 1.3 Mb. We performed an array CGH analysis for 27 NK‐cell lymphoma/leukemia cases that were classified into two disease groups based on the World Health Organization Classification (10 aggressive NK‐cell leukemia cases and 17 extranodal NK/T‐cell [NK/T] lymphomas, nasal type). We identified the differences in the genomic alteration patterns of the two groups. The recurrent regions characteristic of the aggressive NK‐cell leukemia group compared with those of the extranodal NK/T lymphoma, nasal‐type group, were gain of 1q and loss of 7p15.1‐p22.3 and 17p13.1. In particular, gain of 1q23.1‐24.2 (P = 0.041) and 1q31.3‐q44 (P = 0.003–0.047), and loss of 7p15.1‐p22.3 (P = 0.012–0.041) and 17p13.1 (P = 0.012) occurred significantly more frequently in the former than in the latter group. Recurrent regions characteristic of the extranodal NK/T lymphoma, nasal‐type group, compared with those of the other group were gain of 2q, and loss of 6q16.1‐q27, 11q22.3‐q23.3, 5p14.1‐p14.3, 5q34‐q35.3, 1p36.23‐p36.33, 2p16.1‐p16.3, 4q12, and 4q31.3‐q32.1. Our results can be expected to provide further insights into the genetic basis of lymphomagenesis and the clinicopathologic features of NK‐cell lymphomas/leukemias.

Gene expression signatures delineate biological and prognostic subgroups in peripheral T-cell lymphoma.

Peripheral T-cell lymphoma (PTCL) encompasses a heterogeneous group of neoplasms with generally poor clinical outcome. Currently 50% of PTCL cases are not classifiable: PTCL-not otherwise specified (NOS). Gene-expression profiles on 372 PTCL cases were analyzed and robust molecular classifiers and oncogenic pathways that reflect the pathobiology of tumor cells and their microenvironment were identified for major PTCL-entities, including 114 angioimmunoblastic T-cell lymphoma (AITL), 31 anaplastic lymphoma kinase (ALK)-positive and 48 ALK-negative anaplastic large cell lymphoma, 14 adult T-cell leukemia/lymphoma and 44 extranodal NK/T-cell lymphoma that were further separated into NK-cell and gdT-cell lymphomas. Thirty-seven percent of morphologically diagnosed PTCL-NOS cases were reclassified into other specific subtypes by molecular signatures. Reexamination, immunohistochemistry, and IDH2 mutation analysis in reclassified cases supported the validity of the reclassification. Two major molecular subgroups can be identified in the remaining PTCL-NOS cases characterized by high expression of either GATA3 (33%; 40/121) or TBX21 (49%; 59/121). The GATA3 subgroup was significantly associated with poor overall survival (P = .01). High expression of cytotoxic gene-signature within the TBX21 subgroup also showed poor clinical outcome (P = .05). In AITL, high expression of several signatures associated with the tumor microenvironment was significantly associated with outcome. A combined prognostic score was predictive of survival in an independent cohort (P = .004).

Synergistic action of the microRNA-17 polycistron and Myc in aggressive cancer development.

The c13orf25/miR‐17 cluster, which is responsible for 13q31‐q32 amplification in malignant lymphoma, contains the microRNA‐17‐18‐19‐20‐92 polycistron. A previous study demonstrated that this polycistron could modulate tumor formation following transplantation of microRNA 17‐19b into Eu‐myc mice. Another study reported that Myc can upregulate the miR‐17 cluster by binding directly upstream of the miR‐17 locus. These findings suggest that Myc and the miR‐17 cluster synergistically contribute to cancer development. In the study presented here, we observed recurrent 13q31‐32 amplification in MYC‐rearranged lymphomas (11 of 47 cases). Quantitative real‐time polymerase chain reaction analysis of c13orf25 for MYC‐rearranged lymphomas demonstrated that cases with 13q31‐32 amplification showed significantly higher expression of c13orf25 than cases without such amplification, although cases without 13q31‐32 amplification still showed slight upregulation of c13orf25. To investigate the relationship between Myc and the miR‐17 polycistron in tumorigenesis, we engineered rat fibroblasts (Rat‐1) that constitutively express the miR‐17 polycistron (miR), Myc, or both miR and Myc. The highest level of miR expression was detected in Rat‐1 transfected with both miR and Myc, whereas Myc transfectant cells alone also showed slight upregulation of miR. Furthermore, we demonstrated that nude mice injected with Rat‐1 transfected with both miR and Myc presented more accelerated tumor growth than those injected with Myc transfectant cells. These results suggest that miR is stably upregulated in the presence of constitutive expression of Myc, and that the deregulation of miR and Myc synergistically contribute to aggressive cancer development, probably by repressing tumor suppressor genes. (Cancer Sci 2007; 98: 1482–1490)

Gain‐of‐function mutations and copy number increases of Notch2 in diffuse large B‐cell lymphoma

Signaling through the Notch1 receptor has a pivotal role in early thymocyte development. Gain of Notch1 function results in the development of T‐cell acute lymphoblastic leukemia in a number of mouse experimental models, and activating Notch1 mutations deregulate Notch1 signaling in the majority of human T‐cell acute lymphoblastic leukemias. Notch2, another member of the Notch gene family, is preferentially expressed in mature B cells and is essential for marginal zone B‐cell generation. Here, we report that 5 of 63 (~8%) diffuse large B‐cell lymphomas, a subtype of mature B‐cell lymphomas, have Notch2 mutations. These mutations lead to partial or complete deletion of the proline‐, glutamic acid‐, serine‐ and threonine‐rich (PEST) domain, or a single amino acid substitution at the C‐terminus of Notch2 protein. Furthermore, high‐density oligonucleotide microarray analysis revealed that some diffuse large B‐cell lymphoma cases also have increased copies of the mutated Notch2 allele. In the Notch activation‐sensitive luciferase reporter assay in vitro, mutant Notch2 receptors show increased activity compared with wild‐type Notch2. These findings implicate Notch2 gain‐of‐function mutations in the pathogenesis of a subset of B‐cell lymphomas, and suggest broader roles for Notch gene mutations in human cancers. (Cancer Sci 2009; 100: 920–926)