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MEF2D fusion (M-fusion) genes are newly discovered recurrent gene abnormalities that are detected in approximately 5% of acute lymphoblastic leukemia (ALL) cases. We previously found that the loss of the micro RNA target site in wild-type MEF2D gene by translocation led to strong expression of M-fusion protein in ALL cells by evasion from micro RNA and that M-fusion protein inhibited the transcriptional activity of PAX5, a B-cell differentiation regulator, in a dose-dependent manner. These findings prompted us to explore drugs that induced proteolysis of M-fusion protein as possible therapeutic agents for M-fusion-positive ALL. We developed a high-throughput screening system to find compounds that reduced protein expression level of MEF2D. The expression vector of the fusion protein of N-terminal half of MEF2D (MEF2D N) and luciferase (MEF2D N-Luc) was stably transfected to 293T cells (MEF2D N-Luc/293T). Stable transfectant of the expression vector of luciferase was also established (Luc/293T). We could easily measure protein expression level in these cells by luciferase assay. We screened 3766 compounds with known pharmaceutical activities with this system and selected staurosporine, a multi-kinase inhibitor, as a possible proteolysis-inducer of MEF2D. Staurosporine strongly reduced the luciferase value in MEF2D N-Luc/293T but not in Luc/293T (Figure 1A). Staurosporine induced proteolysis of MEF2D-HNRNPUL1 (M-H) and MEF2D-DAZAP1 (M-D) in M-fusion-positive ALL cell lines within 6 h. Proteolysis of M-fusion proteins were inhibited not by MG-132, a proteasome inhibitor, but by Z-VAD FMK, a caspase inhibitor, indicating that these proteolyses were caspase-dependent (Figure 1B). Consistent with this, Z-VAD-FMK blocked apoptosis by staurosporine in M-H positive ALL cell lines . We confirmed the cleavage of M-H by caspase 3 and caspase 7 in vitro and identified the cleavage site (Figure 1C). Furthermore, staurosporine demonstrated stronger cytotoxic effect on M-fusion-positive ALL cell lines than M-fusion-negative ones (Figure 1D). These results indicated that staurosporine induced apoptosis of M-fusion-positive ALL cells through caspase-dependent proteolysis of M-fusion protein at least in part. Luciferase-based proteolysis screening provided a novel strategy for the development of anti-cancer drugs. Figure legends Figure 1. A. Staurosporine strongly reduced the luciferase value in MEF2D N-Luc/293T but not in Luc/293T. MEF2D N-Luc/293T were treated with 3766 compounds (2uM each) for 24 h. Then luciferase assays were performed to estimate the amount of MEF2D N-Luc protein. Top 15 compounds which reduced the relative luciferase value were selected for the second screening where compounds were added to MEF2D N-Luc/293T and Luc/293T, then we estimated their effect on the expression of MEF2D N-Luc and luciferase. Results of the second screening were plotted on a scattergram, on which the relative luciferase value in Luc/293T and MEF2D N-Luc/293T were set on the Y-axis and X-axis, respectively. Relative luciferase values are relative values to those of control cells treated with vehicle (DMSO). Staurosporine and K252a, an analog of staurosporine, were selected as hit compounds which reduced MEF2D N-Luc but not luciferase. B. Staurosporine reduced the expression of M-H and M-D, which inhibited by Z-VAD FMK. M-H-positive ALL cell lines, Kasumi-7 and Kasumi-9 and a M-D-positive ALL cell line, TS-2 were treated with 1uM staurosporine, 20uM Z-VAD-FMK, or both for 6 h. Cells were lysed and subjected to immunoblotting with indicated antibodies. C. Caspase-3 and caspase-7 cleaved M-H in vitro. M-H and its mutants with aspartate substitutions at possible caspase cleavage sites were synthesized with [35S]-methionine-labeled in vitro translation and were incubated with purified caspase-3 or -7. Cleaved fragments were resolved on SDS-PAGE and visualized by autoradiography. Mutant 4 was resistant to the cleavage by both caspases. D. Staurosporine demonstrated stronger cytotoxic effect on M-fusion-positive ALL cell lines than M-fusion-negative ones. ALL cell lines were treated with staurosporine at the indicated doses for 24 h. Cell viabilities were measured using MTT assay. Figure 1 Disclosures Kiyoi: Takeda Pharmaceutical Co., Ltd.: Research Funding; Pfizer Japan Inc.: Honoraria; Astellas Pharma Inc.: Honoraria, Research Funding; Kyowa Hakko Kirin Co., Ltd.: Research Funding; Chugai Pharmaceutical Co., Ltd.: Research Funding; Sumitomo Dainippon Pharma Co., Ltd.: Research Funding; FUJIFILM Corporation: Research Funding; Eisai Co., Ltd.: Research Funding; Bristol-Myers Squibb: Research Funding; Daiichi Sankyo Co., Ltd: Research Funding; Nippon Shinyaku Co., Ltd.: Research Funding; Zenyaku Kogyo Co., Ltd.: Research Funding; Otsuka Pharmaceutical Co.,Ltd.: Research Funding; Perseus Proteomics Inc.: Research Funding.

Although survival rate for children with Acute Lymphoblastic Leukemia (ALL) now exceeds about 90%, the outcome of adult patients with ALL is extremely poor. These differences might be attributed to the lack of insights into pathogenesis and clinical behavior of adult-ALL. Gross chromosomal alterations including chromosome translocations and aneuploidy are considered as early events in ALL and constitute disease subtypes. To identify chromosome translocations underlying adult with Ph-negative B-ALL, we performed RNA-seq analysis on RNA from individuals with B-ALL who had been treated on the Japan Adult Leukemia Study Group (JALSG) ALL202-O protocol (n = 149). We successfully identified chromosome translocations in 100 patients (67.1%). ZNF384 fusions were most frequently detected in 30 patients (20.1%) and they had wide range of fusion partners. DUX4- and MEF2D- fusions were also recurrently found in 7 (4.7%) and 9 (6.0%) patients, respectively. Chromosome translocations activating kinase and cytokine receptor were found in 25 patients (16.8%) with Ph-like gene expression profile. These alterations were almost completely mutually exclusive indicating these are likely to be primary genetic events. For simplicity, here we define (1) fusions involving ZNF384, DUX4, MEF2D, CEBP and PAX5 as well as TCF3-PBX1 and ETV6-RUNX1 as Transcription Factor fusions (TF fusions; 49% of patients), (2) fusions involving CRLF2, JAK2, PDGFRB, EPOR and ABL as Kinase/cytokine-receptor Activating fusions (KA fusions; 15%) and (3) non-recurrent fusions or the absence of fusions/aneuploidy as B-others (30%). First, we analyzed impact of the patient age on types of fusion genes, based of combined data of ALL202-O cohort, childhood B-ALL cohort (Lilljebjörn H, et al. 2016: n = 189) and ALL202-U cohort (Yasuda T, et al. 2016: n = 54). We found that incidence of ZNF384-, CEBP- fusions and B-others increases as patients age, whereas ETV6-RUNX1 and PAX5 fusions were more prevalent in younger patients, exhibiting negative association with age. DUX4 fusions and TF fusions were most prevalent in Adolescent and Young Adult (AYA) generation. JAK2-, PDGFRB-, EPOR- and KA- fusions were positively correlated with age. Next, we analyzed association between patient survival and types of fusions. In Japanese adult B-ALL cohort (ALL202-O and ALL202-U cohort), we observed ZNF384-, DUX4- fusions and TCF3-PBX1 were associated with better disease-free survival than B-others. Furthermore, when combined, MEF2D- (n = 14), CEBP- (n = 4), PAX5- fusions (n = 2) and ETV6-RUNX1 (n = 2) exhibited significantly better disease-free survival than B-others, indicating TF fusions were associated with an improved outcome. In contrast, KA fusions were associated with poorer disease-free survival than B-others. KMT2A fusions were comparable with B-others regarding to patient disease-free survival. These results allowed us to develop a prognostic schema to identify three distinct risk profile groups, based on types of fusion genes and cytogenetics (Table1); favorable-risk (5-year rate of disease-free survival 67.4%), intermediate-risk (5-year rate of disease-free survival 42.5%) and adverse-risk (5-year rate of disease-free survival 9.6%). This prognostic schema predicted the outcome independently of age, sex and methotrexate dose in multivariate analysis (p < 0.001). In conclusion, we promoted a better understanding of the genetic basis of adult B-ALL by focusing on fusion genes. Each chromosome translocations were closely associated with age. ZNF384-, KA fusions and B-others were characteristic for older-adult patients (40-65 years old) with B-ALL. We clearly demonstrated specific primary chromosome abnormalities are strong prognostic marker. Functional properties of primary genetic events (TF fusions vs. KA fusions) might be a key determinant of biological characteristics and clinical outcome. Disclosures Kiyoi: Novartis Pharma K.K.: Research Funding; Celgene Corporation: Research Funding; Zenyaku Kogyo Co., Ltd.: Research Funding; FUJIFILM Corporation: Research Funding; Chugai Pharmaceutical Co., Ltd.: Research Funding; Bristol-Myers Squibb: Honoraria; Otsuka Pharmaceutical Co., Ltd.: Research Funding; Takeda Pharmaceutical Co., Ltd.: Research Funding; Sanofi K.K.: Research Funding; Nippon Shinyaku Co., Ltd.: Research Funding; Kyowa Hakko Kirin Co., Ltd.: Research Funding; Sumitomo Dainippon Pharma Co., Ltd.: Research Funding; Astellas Pharma Inc.: Research Funding; Phizer Japan Inc.: Research Funding; Eisai Co., Ltd.: Research Funding. Naoe:Astellas Pharma Inc.: Research Funding; Fujifilm Corporation: Patents & Royalties, Research Funding; Nippon Shinyaku Co., Ltd.: Research Funding; Otsuka Pharmaceutical Co., Ltd.: Research Funding; Pfizer Japan Inc.: Research Funding; Toyama Chemical Co., Ltd.: Research Funding.

ZNF384 fusion (Z-fusion) genes are recently identified recurrent fusion genes of B-acute lymphoblastic leukemia (ALL) and cause differentiation block of B-cells; however, its molecular mechanisms have yet to be clarified. Common structural character of Z-fusion proteins is that fusion partners are fused to the N-terminal end of full-length ZNF384 (Figure 1A), suggesting that protein-fusions confer specific transcriptional targets on ZNF384. We searched Z-fusion-specific transcriptional targets that could cause differentiation block of B-cells by analyzing the data of gene expression profile of 54 primary B-ALL samples containing 9 Z-fusion positive ALL. We selected ID2 and SALL4 as potential targets. Both genes were expressed markedly higher in Z-fusion-positive ALL. ID2 acts as an inhibitor of E2A, B cell differentiation regulator, and SALL4 plays essential roles in maintaining pluripotency of embryonic stem cells. In the luciferase assays, EP300-ZNF384 (E-Z) and SYNRG-ZNF384 (S-Z) showed stronger transcriptional activities on the promoters of these genes than wild-type ZNF384 (Wild-Z). The introduction of E-Z or S-Z into 293T cells and THP-1 cells induced mRNA expression of these genes more strongly than that of Wild-Z (Figure 1B). We identified Z-fusion binding sites in the promoters of these genes. DNA binding abilities of Z-fusions to these sites were not stronger than that of Wild-Z in electro mobility shift assay. GST-pull down assay showed that E-Z associated with EP300 more strongly than Wild-Z (Figure 1C). Consistent with this, co-expression of EP300 enhanced the transcriptional activity of E-Z better than that of Wild-Z (Figure 1D). These results indicated that ID2 and SALL4 were Z-fusion-specific transcriptional targets and that the high affinity to EP300 was responsible for the strong transcriptional activity of Z-fusions. Our results shed a new insight into the molecular mechanisms of leukemia development by Z-fusions. Figure legends Figure 1. A. Schematic presentation of structures of Wild-Z and Z-fusion proteins. B. Introduction of Z-fusion genes enhanced the mRNA expression of ID2 and SALL4. Wild-Z and Z-fusion genes were introduced to THP-1 cells by nucleofection. Twenty-four hours after gene introduction, the mRNA expression of ID2 and SALL4 were quantified by RQ-PCR and potted on bar charts as relative values to the mRNA expression in the control cells. The expression of ID2 and SALL4 were shown in the left and right bar charts, respectively. C. E-Z associated with HAT more strongly than Wild-Z. Glutathione beads attached with Glutathione S transferase (GST) or GST-fused HAT were incubated with Wild-Z or E-Z synthesized in vitro with [35S]-Methionine labeling. Wild-Z or E-Z associated with GST-HAT were visualized with autoradiography (left panel), quantified, and plotted on the bar charts (right panel). Of note, the quantified association were adjusted for the ratio of the number of methionine containing in Wild-Z and E-Z, 12 to 45, and plotted on the bar charts. D. Co-expression of EP300 enhanced the transcriptional activity of E-Z better than that of Wild-Z. Luciferase assay were performed with or without co-expression of EP300 and the relative value to the control were plotted on the bar charts. Disclosures Naoe: Astellas Pharma Inc.: Research Funding; Fujifilm Corporation: Patents & Royalties, Research Funding; Nippon Shinyaku Co., Ltd.: Research Funding; Otsuka Pharmaceutical Co., Ltd.: Research Funding; Pfizer Japan Inc.: Research Funding; Toyama Chemical Co., Ltd.: Research Funding. Kiyoi:FUJIFILM Corporation: Research Funding; Eisai Co., Ltd.: Research Funding; Astellas Pharma Inc.: Research Funding; Kyowa Hakko Kirin Co., Ltd.: Research Funding; Sumitomo Dainippon Pharma Co., Ltd.: Research Funding; Chugai Pharmaceutical Co., Ltd.: Research Funding; Otsuka Pharmaceutical Co., Ltd.: Research Funding; Zenyaku Kogyo Co., Ltd.: Research Funding; Novartis Pharma K.K.: Research Funding; Nippon Shinyaku Co., Ltd.: Research Funding; Celgene Corporation: Research Funding; Bristol-Myers Squibb: Honoraria; Takeda Pharmaceutical Co., Ltd.: Research Funding; Phizer Japan Inc.: Research Funding; Sanofi K.K.: Research Funding.

Diffuse large B-cell lymphoma (DLBCL) comprises molecularly distinct subgroups such as activated B-cell-like (ABC) and germinal center B-cell-like (GCB) DLBCLs. We previously reported that CD5(+) and CD5(-)CD10(+) DLBCL constitute clinically relevant subgroups. To determine whether these 2 subgroups are related to ABC and GCB DLBCLs, we analyzed the genomic imbalance of 99 cases (36 CD5(+), 19 CD5(-)CD10(+), and 44 CD5(-)CD10(-)) using array-based comparative genomic hybridization (CGH). Forty-six of these cases (22 CD5(+), 7 CD5(-)CD10(+), and 17 CD5(-)CD10(-)) were subsequently subjected to gene-expression profiling, resulting in their division into 28 ABC (19 CD5(+) and 9 CD5(-)CD10(-)) and 18 GCB (3 CD5(+), 7 CD5(-)CD10(+), and 8 CD5(-)CD10(-)) types. A comparison of genome profiles of distinct subgroups of DLBCL demonstrated that (1) ABC DLBCL is characterized by gain of 3q, 18q, and 19q and loss of 6q and 9p21, and GCB DLBCL is characterized by gain of 1q, 2p, 7q, and 12q; (2) the genomic imbalances characteristic of the CD5(+) and CD5(-)CD10(+) groups were similar to those of the ABC and GCB types, respectively. These findings suggest that CD5(+) and CD5(-)CD10(+) subgroups are included, respectively, in the ABC and GCB types. Finally, when searching for genomic imbalances that affect patients' prognosis, we found that 9p21 loss (p16(INK4a) locus) marks the most aggressive type of DLBCL.

Transcription factor GATA-2 is implicated in the survival and growth of multipotential progenitors. Here we report that the promyelocytic leukemia zinc finger (PLZF) protein can interact with GATA-2 and can modify its transactivation capacity. Fanconi anemia zinc finger (FAZF), a PLZF-homologous protein that has been variously described as ROG (repressor of GATA), and TZFP (testis zinc finger protein) also interact with GATA-2. The zinc finger region of GATA-2 is required for binding to PLZF and FAZF, but distinct interfaces on the PLZF and FAZF molecules mediate the interaction, suggesting that GATA-2 activity is controlled by these 2 homologous proteins through distinct mechanisms. GATA-2 can also physically associate with the PLZF-RARα fusion protein generated by the t(11;17) chromosomal translocation associated with acute promyelocytic leukemia (APL). Functional experiments showed that this interaction has the capacity to render GATA-dependent transcription responsive to treatment with a combination of all-trans retinoic acid and the histone deacetylase inhibitor trichostatin A (TSA). This combination of drugs has been shown to stimulate the terminal differentiation of leukemic t(11;17)-associated APL blasts, raising the possibility that GATA target genes may be involved in the molecular pathogenesis of APL.

We report here the results of polymerase chain reaction (PCR) for bcr- abl transcript and clinical details derived from 64 chronic myelogenous leukemia (CML) patients after allogeneic bone marrow transplantation (BMT). A total of 139 samples (2 to 220 weeks after BMT) were analyzed and bcr-abl transcript was detected in 99 samples from 52 patients. Patients were defined as bcr-abl early negative (EN) if they had > or = 1 negative PCR result < or = 1 year post-BMT (n = 13), and bcr-abl late positive (LP) if they had > or = 1 positive PCR result > or = 1 year post-BMT (n = 21). Among LP patients, only two patients had hematologic/cytogenetic (clinical) relapse. Another 19 LP patients remained in clinical remission 7 to 130 weeks after positive analysis for bcr-abl transcript, including 5 patients who had persistent bcr-abl transcript detectable even 2 years after BMT. To estimate the relationship between clinical data and residual bcr-abl transcript, EN patients are compared with LP patients. However, no clinical data studied were significantly associated with the persistent PCR positivity. If only patients in chronic phase are compared, the t-test showed significant correlation between leukocyte count just before BMT and sustained bcr-abl transcript (P < .05). These results suggest that PCR positivity is frequently observed in CML patients who sustain clinical remission after BMT, without being predictive of imminent clinical relapse. Tumor burden at the time of BMT may play an important role in the latency of bcr-abl positivity after BMT.

PAX5 is a transcription factor required for B-cell development and maintenance. We previously showed that PAX5-PML, a fusion gene found in acute lymphoblastic leukemia (ALL), dominant negatively inhibited PAX5 transcriptional activity. Reported data including ours revealed that PAX5 fusion proteins had possible oncogenic ability; however, leukemogenicity of PAX5 fusion genes and other PAX5 mutations in mice model has not been clarified, yet. Here we demonstrated leukemia development in mice by introducing PAX5-PML. Pro B cells derived from mouse fetal liver were transfected with PAX5-PML expression vector and transplanted into mice. All 8 transplanted mice died with pro B ALL from day 63 to 158. Leukemic cells could be serially transplanted and mice died more rapidly with repetition (Figure A). Among the target genes transcriptionally activated by PAX5, expressions of BLNK, Fcer2a, and CD72 were significantly repressed in leukemia cells but repression of CD19 and CD79a were mild, suggesting the importance of down regulation of these genes for differentiation block. We compared mRNA expression profile between leukemia cells and normal pro B cells and gene set enrichement analysis (GSEA) identified candidates for second hits for development of leukemia. We analyzed the mechanism of the selective repression of CD19, Fcer2a, and BLNK and the significance of the second hit candidates, using a cell line established from leukemia cells of the third transplanted mouse. The results will show the meeting. Figure 1 Figure 1. Disclosures Sugimoto: Otsuka Pharmaceutical Co., Ltd: Employment. Naoe:Zenyaku Kogyo: Research Funding; Dainippon Sumitomo Pharma: Research Funding; Kyowa Hakko Kirin Co. LTD: Research Funding; Chugai Pharmaceutical Co. LTD: Research Funding; Novartis Pharma,: Research Funding; Bristol-Myers Squibb: Research Funding; Otsuka Pharmaceutical Co. LTD: Research Funding; FUJIFILM Corporation: Research Funding. Kiyoi:Zenyaku Kogyo: Research Funding; Dainippon Sumitomo Pharma: Research Funding; Kyowa Hakko Kirin Co. LTD.: Research Funding; Chugai Pharmaceutical Co. LTD: Research Funding; Bristol-Myers Squibb: Research Funding; FUJIFILM Corporation: Research Funding.

Introduction: Peripheral T-cell lymphomas (PTCLs) are a heterogeneous group of non-Hodgkin lymphomas noted for their poor prognosis. Their molecular pathogenesis has not been entirely elucidated. We previously found that primary thyroid T-cell lymphoma (PTTL) is a distinct entity among heterogeneous PTCLs and that this disease is characterized by the genomic loss of 6q24 (Br J Haematol., 161:214-223). In this study, we extended the analysis to other types of PTCLs and performed functional assays to identify causative genes located on 6q24. Methods: Focusing on chromosome 6q loss, we reexamined previous comparative genomic hybridization data from 267 PTCL cases comprising 6 PTTLs, 51 PTCLs-not otherwise specified (NOS), 62 adult T-cell leukemias/lymphomas, 35 natural killer (NK)-cell lymphomas, 39 angioimmunoblastic T-cell lymphomas (Genes Chromosomes Cancer, 46:37-44), and 74 anaplastic large cell lymphomas (Br J Haematol., 140:516-526). Gene expression levels were determined by using published gene expression profiling (GEP) data (GSE6338 and GSE19069) and quantitative real-time reverse transcription polymerase chain reaction (RT-PCR). Subsequently, we established Tet-Off cell lines belonging to several lineages (6 T-cell lines, 1 NK-cell line, 4 B-cell lines, 1 myeloid cell line, and 3 epithelial cell lines) for functional analyses. Results: Genomic loss of 6q24 was observed in 8% (n = 267) of PTCL cases, and it occurred most frequently in PTTL cases (67%; n = 6). All the genomic losses were heterozygous; homozygous loss of this region was not observed in our analysis. The smallest region of deletion, observed in a PTTL case, was considered the minimal common region (MCR) of 6q24 loss. The MCR contained 2 known coding genes, STX11 and UTRN. Combined GEP data and quantitative RT-PCR analyses showed that the expression of STX11, but not UTRN, was markedly lower in PTCL than in normal T-cells. We therefore regarded STX11 as the most probable candidate gene located in 6q24. Syntaxin 11, encoded by STX11, is a t-SNARE protein that plays a role in binding vesicles to cell membranes, and alteration of STX11 in the germline causes familial hemophagocytic syndrome type 4. To further evaluate genomic alteration of STX11, mutation analysis was performed on PTCL-NOS and PTTL cases as well as T-cell lines, for which adequate DNA was available. This revealed STX11 mutations in 2 cases (1 PTCL-NOS case and 1 T-cell line). Wild-type STX11 expression suppressed the proliferation of T-cell lines bearing genomic alterations at the STX11 locus only, and it did not show suppressive effects on other lineage cell lines (Fig. 1). Expression of STX11 induced cellular apoptosis in the cell line, although the number of apoptotic cells induced was relatively small. Interestingly, expression of a novel STX11 mutant (p.Arg78Cys), observed in a T-cell line, did not exert suppressive effects on the induced cell lines suggesting that there was a loss-of-function mutation (Fig. 2). Finally, we evaluated the clinical impact of STX11 alteration in PTTL and PTCL-NOS cases where data were available. This showed that PTCL-NOS cases with genomic alterations of STX11 tended to have a poorer prognosis than those without (Fig. 3; P = 0.069). Conclusion: In the present study, we examined the MCR of 6q24 loss and showed that STX11 acts as a tumor suppressor gene in PTCLs only. These findings provide a novel approach for understanding the molecular pathogenesis of PTCLs, and they may contribute to the future development of new drugs for the treatment of PTCLs. Disclosures No relevant conflicts of interest to declare.

Abstract 416 In B-cell lymphomas, several chromosomal translocations are associated with specific histological subtypes. CCND1/IGH is detected in more than 90 % of the cases with mantle cell lymphoma (MCL), and BCL2/IGH is characteristically observed in follicular lymphoma and diffuse large B-cell lymphoma (DLBCL) of germinal center (GC) B-cell origin. Although these strong correlations are clinically recognized, their biological mechanism is not clearly explained so far. According to the results of translocation breakpoint mapping, both CCND1 and BCL2 translocations are considered to be generated by an error during physiological VDJ rearrangement of the IGH gene in the precursor-B cell stage. We hypothesized that the occurrence of these translocations in the immature B-cell stage, probably as an initial genetic event, should have a special impact on the determination of resulting lymphoma histology. We generated a mouse model mimicking human lymphoma with CCND1 or BCL2 translocation by lentivirally introducing these genes into Tp53+/− B6 mouse bone marrow cells and transplanting them to lethally irradiated wild-type B6 mice. In this model, CCND1 or BCL2 is expressed from immature to mature B cell stage under the control of the CD19 promoter, and subsequently mutations accumulate in the background of Tp53 haploinsufficiency. Both mice developed B-cell lymphomas several months after the transplantation, but their tumors showed some different features. CCND1-Tp53+/− mice developed B220lowCD5+CD23− tumors that expand into the B-cell area with sparing the GC, whereas BCL2-Tp53+/− mice preferentially developed B220highCD5−CD23+ tumors that tend to localize in the GC. Additionally, somatic hypermutation (SHM) analysis of the IGH gene of the tumor cells revealed obviously higher mutation frequency in the BCL2-Tp53+/− mice than in the CCND1-Tp53+/- mice (p There has been a debate whether B-1 cell population exists in humans, but it is recently proposed that the phenotype of human B-1 cells is CD20+CD27+CD43+CD70− by testing sort-purified B cell fractions for fundamental B-1 cell functions based on mouse studies (J Exp Med 2011;208:67–80). Interestingly, flow cytometric analysis of the human MCL tumor cells has shown that they mostly express this provisional B-1 cell phenotype, supporting the idea that human MCL is also derived from B-1 cells. Our mouse model precisely reproduces the link between CCND1 and BCL2 translocations and the resulting lymphoma subtypes in humans. It is assumed that these translocations trigger the cell expansion of different B-cell subgroups, which consequently leads to the development of lymphoma of distinct histology. Our findings provide new insights into the mechanism of lymphoma subtype determination. Disclosures: No relevant conflicts of interest to declare.

Abstract 3448 Age-related EBV-associated B-cell lymphoproliferative disorder (AR-EBLPD) is classified as a subtype of diffuse large cell lymphoma (DLBCL) according to the WHO classification. However, molecular genetic characterization of AR-EBLPD remains largely unknown. We studied expression profiles of 5 AR-EBLPD and 8 EB-negative DLBCL samples using the Agilent 44K human oligonucleotide microarray. Total RNA was extracted from fresh-frozen tumor samples. Each microarray slide was converted into datasets using the Agilent Micro Array Scanner and Feature extractions. Data was standardized with Z-scores. Differences in mRNA expression levels between two sample groups were calculated using a two-sided t-test. A total of 1973 probes showed a p-value less than 0.05 with less than a 25% false discovery rate (FDR). These probes included 1688 genes. The number of probes showing high expression in AR-EBLPD and EB-negative DLBCL was 804 (693 genes) and 1169 (995 genes), respectively. First, we selected the top 300 differentially expressed genes. Genes highly expressed in AR-EBLPD included IL6, TNFAIP3, HOPX, and SLAMF1. IL6 is known as a gene encoding a cytokine which functions in inflammation and the maturation of B lymphocytes, and TNFAIP3 is known as a negative regulatory gene of the NF-kB pathway. HOPX and SLAMF1 are reported as genes related to lymphocyte function or the immune system (Schwartzberg et al. Nature immunology 2009, Hawiger et al. Nature immunology 2011). For better characterization, we next performed Gene Ontology Analysis using the WEB-based GEne SeT AnaLysis Toolkit and found that categories of external stimulus and inflammatory responses were enriched in AR-EBLPD. The Kyoto Encyclopedia of Genes and Genomes (KEGG)-signaling analyses showed that pathways of the NOD-like receptor (p-value =1.30e-06), JAK-STAT (p-value =9.01e-06), and Toll-like receptor (p-value =0.0002) were characteristic of AR-EBLPD. These results implied that inflammation would be prominent in AR-EBLPD cases. For validation, we next performed Gene Set Enrichment Analysis (GSEA) using all the database of KEGG pathways (186 gene sets). Dominant gene sets in AR-EBLPD included the cytokine-cytokine receptor interaction [Normalized Enrichment Score (NES) =2.66, p-value (Figure 1) Gene Set Enrichment Analysis of 5 AR-EBLPD and 8 EB-negative DLBCL samples. The NF-kB signature reported from an NIH group (Puente et al. Nature 2011) was enriched in AR-EBLPD [Normalized Enrichment Score (NES) =2.20, p-value Disclosures: No relevant conflicts of interest to declare.

BCL2/IGH translocation is a hallmark of follicular lymphoma and diffuse large B-cell lymphoma of germinal center B-cell type. Although being a strong determinant of these histological subtypes, this translocation is considered to be insufficient by itself and further gene alterations are necessary for cellular transformation. In Eμ-BCL2 transgenic (Tg) mice, B-lineage cells are increased by several-fold compared to wild-type (WT) mice, but only 5–15 % of them develop disease in the first year of life. To clarify how the BCL2 translocation contributes to the development of specific lymphoma subtypes, we created two types of chimeric mouse models to characterize the biological features of BCL2-overexpressing B cells in normal individuals. First, we introduced CD19 promoter-driven BCL2 and its mutant genes to a minor population of murine bone marrow cells by using a lentiviral vector system and transplanted into irradiated mice. BCL2-overexpressing B cells showed increased follicular and reduced marginal zone populations. The same phenotypic shift was observed in B cells introducing BCL2-Y28F mutant that retained anti-apoptotic function, but a defective mutant BCL2-G142A and a mock vector did not affect B-cell phenotype. Additionally, BCL2-introduced B cells showed decreased cell size compared to those introduced BCL2-G142A and mock vectors. To assess the functional alteration of BCL2-overexpressing B cells, TNP-Ficoll binding experiment was performed. The result showed diminished T-cell independent response in parallel with decreased marginal zone B cells. The low transformation frequency of B cells in Eμ-BCL2 Tg mice has been partly explained by their propensity to reside in the G0 phase of the cell cycle (reviewed in Oncogene, 18:5268,1999). We hypothesized that the microenvironment of B cells in Eμ-BCL2 Tg mice might be altered by abnormal B cells themselves. To evaluate the influence of the different microenvironments on BCL2-overexpressing B cells, we next made Eμ-BCL2/CAG-GFP double Tg mice and transferred their bone marrow mononuclear cells into WT or Eμ-BCL2 Tg mice. Blastic cell population of BCL2+GFP+ B cells was larger in those transferred to WT mice compared to those transferred to Eμ-BCL2 Tg mice, regardless of the same phenotypic preference toward follicular B cells. BrdU uptake experiments demonstrated continuous cell cycle progression of the BCL2+GFP+ B cells in WT mice but repressed cell cycle of those in Eμ-BCL2 Tg mice. In immunohistochemical analysis, splenic follicles were disorganized with reduced follicular dendritic cells and inadequate T cell accumulation in Eμ-BCL2 Tg mice. Functional impairment of splenic follicles in Eμ-BCL2 Tg mice might be caused by decreased marginal zone B cell subset, as the antigen capture and delivery by marginal zone B cells was reported to play an important role in the development of follicular dendritic cells. To understand the fate of BCL2-overexpressing B cells after stimulation, we finally assessed their terminal differentiation capacity in vitro. Plasma cell differentiation was suppressed in B cells derived from Eμ-BCL2 Tg mice under either LPS or anti-IgM antibody stimulation. BCL2 is reported to impede the activity of transcription factor NF-AT (Proc Natl Acad Sci93:9545,1996; Nature386:728,1997), and we found that calcineurin inhibitor FK506 suppressed plasma cell differentiation of WT B cells. Gene regulation patterns of the Eμ-BCL2+ B cells were similar to B cells stimulated in the presence of FK506 as well, suggesting that repressed terminal differentiation in Eμ-BCL2+ B cells was partly caused by the suppressed activity of NF-AT. In summary, BCL2-deregulated B cells preferentially differentiate into follicular B cells, and as a result of decreased terminal differentiation in addition to their anti-apoptotic property, they may be obliged to survive and recirculate as memory B cells, and accumulate genetic abnormalities while they repeatedly pass through the germinal center. As the germinal center is the particular site where they can counterbalance the cell cycle-retarding effect of BCL2, it may be a specific place for generating lymphoma triggered by BCL2/IGH translocation. Our results emphasize the importance of the microenvironment of pre-malignant cells during transformation process, and suggest that a simple transgenic mouse model may not be always appropriate for the study of oncogenesis.

Acute-type Adult T-cell leukemia/lymphoma (ATLL) is classified into four clinical subtypes (Acute, Lymphoma, Chronic and Smoldering). Acute-type ATLL comprises clinically heterogeneous groups because this subtype is defined as a group not classified into three other subtypes. When lymphoma-type progress to show PBL involvement of more than 1%, it becomes acute-type ATLL. We tentatively designate such cases showing leukemic cells in peripheral blood with prominent nodal involvement as “acute-type ATLL-PNI”. Clarification of the molecular basis for clinical heterogeneity of Acute-type ATLL is an important issue for therapeutic strategy. We previously studied acute-type ATLL with fulminating leukemic manifestation (more than 60% leukemic cells in peripheral blood) by array CGH and identified a part of the molecular basis for the ATLL subtype (Oshiro et al., Blood107:4500,2006). Here we analyzed six such cases of “acute-type ATLL-PNI” by means of array CGH consisting of 2304 artificial chromosome clones that cover the whole genome at a 1.3 mega base resolution. Regions of recurrent gain were detected on 2p16.1, 3p26.3-26.1, 3p24.2-24.1, 3p22.2-22.1, 3p14.3, 3p14.2-3q13.3, 3q25.32-26.2, 3q26.31-26.33, 3q27.3-29, 6p25.3-24.1, 7q32.3-33 and 17q23.3-24.2 and regions of recurrent loss on 4q21.22-21.23, 6q13-16.3 and 13q32.1-33.1. We compared the genomic profile of these six cases of “acute-type ATLL-PNI” with that of 49 lymphoma-type ATLL cases previously reported by us (Oshiro et al., Blood, 2006). They were very similar to each other and thirteen of the 15 regions of recurrent genomic imbalance in “acute-type ATLL-PNI” were also found in lymphoma-type ATLL (86.7%). Our findings clearly showed that “acute-type ATLL-PNI” have genomic imbalances in common with lymphoma-type ATLL rather than acute-type. Our array data also indicated that “clinically defined” acute-type ATLL contained leukemic-type and lymphoma-type with leukemic transformation.

Marginal zone B cell lymphoma (MZBCL) occupies a distinct disease entity in WHO classification, and it has been suggested that recurrent chromosomal aberrations, such as t(11;18)(q21;q21), t(14;18)(q32;q21), and t(1;14)(p22;q32) are associated with its lymphomagenesis. However, the frequency of chromosomal alteration in marginal zone B cell lymphomas varies according to the anatomical region, and genetic abnormalities other than recurrent translocations involved in the pathogenesis of MZBCL have not yet been fully identified. In this study, we aimed to investigate genomic aberrations in ocular MZBCL and to compare them with those of tumors from other anatomical sites by using genome-wide array comparative genomic hybridization (CGH). The study population comprised 24 cases of primary ocular MZBCL, 11 of pulmonary MZBCL, and seven of nodal MZBCL. FISH analysis for MALT1 gene alteration was performed for ocular and nodal MZBCL and RT-PCR for the detection of API2-MALT1 transcript was performed for pulmonary MZBCL. The recurrent genomic alterations of ocular MZBCL were losses of chromosomes 6q23.3(9/24, 38%), and gains of 3(9/24, 38%), 15(4/24, 16%), 18q(4/24, 16%). T(11;18)(q21;q21) was not detected. The genomic alteration of pulmonary MZBCL included recurrent loss of 13q34 (2/11, 19%). Fusion transcript of t(11;18)(q21;q21) was detected in five out of eight cases (63%). Nodal MZBCL showed neither recurrent genome alteration nor any change in MALT1 gene copy number. In conclusion of genome-wide array CGH analysis, the array CGH profile of ocular MZBCL is distinct from those of pulmonary and nodal MZBCL. The novel finding was recurrent deletion in the 2.9 Mb region at chromosome band 6q23.3–q24.1 including homozygous loss in ocular adnexal marginal zone B cell lymphoma. These findings suggest that deletion of chromosome band 6q23.3–24.1 may constitute a crucial genetic alteration in the lymphomagenesis of ocular MZBCL. For a further examination, we used contig bacterial artificial chromosome (BAC) array CGH, containing the 24 BAC clone to cover the 2.9 Mb region, to analyze nine cases with chromosome band 6q23.3–q24.1 loss. We narrowed the minimal common region down to a length of 586 kb with two genes and four expressed sequence tags (ESTs). All of these genes and ESTs were subjected to RT-PCR and real-time quantitative RT-PCR, and correlation between genomic loss and expression level was found only in TNFAIP3. We thus concluded that the TNFAIP3 gene is the candidate gene for this deleted region. TNFAIP3 is an inhibitor of NF-kB signaling so that loss of this gene may result constitutive activation of NF-kB which was also implicated in MALT lymphomas with t(11;18)(q21;q21) or t(14;18)(q32;q21). Thus, TNFAIP3 may act as a tumor suppressor gene in ocular adnexal marginal zone B cell lymphoma.

Malignant lymphoma frequently shows genomic amplification of 13q31-32, which harbor c13orf25, a host of mature microRNA-17, 18, 19a, 19b, 20a, and 92–1 (Ota et al., Cancer Res 2004). It has been reported that Eμ-myc mice enforced by over-expression of the miR-17-19b, could develop tumor significantly earlier than those without the expression (He et al., Nature 2005), and that overexpression of a proto-oncogene, c-MYC can up-regulate miR-17 cluster via binding directory upstream of miR-17–92 locus (O’ Donnell et al., Nature 2005). These findings suggest that the miR-17 cluster and c-MYC synergistically contribute to cancer development. However, it is still unclear how c-myc acts synergistically in tumorigenesis with the miR-17 cluster, and which genes are essentially regulated by the miR-17 cluster. In this study, we inserted 674 or 755 bp fragments spanning a given miRNA-genomic region in a modified PMXs vector such that they are placed under the control of a LTR promoter, and thus established Rat-1 and NIH3T3 cells which stably expressed miR-17–18-19a–20a-19b-92 or miR-17–18-19a-20-19b (miR-17–92/miR-17-19b). We demonstrated that the miR-17 cluster is highly upregulated under the constitutive expression of c-myc, and that a rat fibroblast (Rat-1) stably expressing both the miR-17 cluster and c-myc (miRs+myc) shows a full-blown neoplastic transformation phenotype. Because the transformation of Rat-1 cells could be due to the suppression of tumor suppressor gene(s), we next conducted screening for predicted tumor related gene(s) by a Western blot analysis with lysate from Rat-1 transfectants. Our examined predicted tumor suppressor genes of miR-17 cluster were p130 (RBR2), PTEN, SOSC-3, Smad2, MeCp2, and TGF-β type II receptor (TβRII). Among these, we could detect significant reduced expression of TβRII protein in the miRs+myc transfectant, although no other predicted targets showed such a significant difference. The expression levels of TβRII gene in Rat-1 and NIH3T3 transfected with miRs+myc were also repressed in comparison to those of the other transfectants. These results demonstrate that a combined effect of RNA destruction and translation inhibition is used by the miR-17 cluster to silence TβRII. To further substantiate TβRII as a direct target of miR-17 cluster, we cloned its 3′UTR sequence downstream of the firefly luciferase gene (pGL3-TβRII). As a control, a construct with the mutated target sequence was also made. Transfection experiments showed a significant repression of the luciferase activity in the wild type construct in comparison to mutated TβRII, thus suggesting that TβRII is direct downstream target of the miR-17 cluster. Up-regulation of miR-17 cluster and c-myc might contribute to tumorigenesis via the down regulation of both the TβRII-Smads and the c-myc-Miz-1-p15INK4b pathways, respectively. Furthermore, we demonstrated that aggressive B-cell lymphoma frequently represents both c-MYC rearrangement and the upregulation of the miR-17 cluster via 13q31 amplification. These results suggest that the deregulation of the miR-17 cluster and c-MYC synergistically contribute to the aggressive clinicopathological features of cancers.

Peripheral T-cell lymphoma, unspecified (PTCL-U) is the most common group among Peripheral T-cell lymphomas (PTCLs). This category consists of the cases which do not belong to any of the recognizable subtypes of PTCLs in WHO classification. PTCL-U comprises heterogeneous groups in morphology and phenotype. Molecular basis of clinical heterogeneity is needed to identify distinct subgroups with clnical relevance. Several reports of conventional cytogenetic studies including comparative genomic hybridization (CGH) showed some recurrent aberrations, but failed to identify the genetic hallmarks to categorize distinct subgroups. So far, no array-based comparative genomic hybridization (array CGH) study for PTCL-U has been reported. Here we analyzed 29 cases of PTCL-U by means of array CGH consisting of 2265 artificial chromosome clones that cover the whole genome at a 1.3 mega base resolution. The analysis clearly divided these cases into two distinct subgroups on the basis of frequency of genomic alterations. One group consists of 17 cases which showed significant lower copy number changes (average copy number gains: 0.5 regions, average copy number losses: 0.1 regions). The other group had average copy number gains of 15.7 regions and losses of 15.0 regions in 12 cases. We designate the former as “simple type” and the latter as “complex type”. In the complex type, regions of recurrent (>20%) gain are detected on chromosome 1q23.3-24.2, 3q25.31-tel, 4p15.1-16.1, 4q28.3-31.23, 5q34, 6p24.1-25.1, 7p21.3-tel, 7p21.1, 7q, 8q24.23, 11q13.4-tel, 12p11.21-11.22, 16p12.3-13.3, 17q11.2-22. Regions of recurrent (>20%) losses are detected on chromosome 1p13.1-13.3, 2q37.3, 4q21.21-21.23, 4q34.3-35.1, 5q21.2-23.1, 6p12.1-q14.3, 6q23.2-24.1, 6q25.1-26, 7p14.3-22.1, 9p21.3, 10p14-qtel, 12p13.1-13.2, 13, 14q12, 16q, 17p, 18p, 20q13-2, 22q11.21-12.2. Median age is 62 years in the simple type and 73 years in the complex type, respectively. Median survival is 27 months in the simple type and 11 months in the complex type. Log-rank test for overall survival between the simple type and the complex type showed inferior survival for the complex type but significance was marginal (p=0.21). Our findings showed that PTCL-U comprised two genetically distinct subgroups, implying that distinct mechanisms underlay in molecular pathogenesis of PTCL-U. Furthermore cilinicopathological features of each group are also being studied.

TEL (ETV6)-AML1 (RUNX1) chimeric gene fusion is the most common genetic abnormality in childhood acute lymphoblastic leukemias (ALL). Evidence suggests that this chimeric gene fusion usually occurs in utero during fetal hematopoiesis and most probably constitutes an initiating or first-hit mutation that is necessary but insufficient for the development of overt, clinical leukemia. In a search for additional secondary and postnatal genetic events that could be linked to leukemia development, we applied a genome- wide array- CGH technique to 24 TEL-AML1 leukemia samples and two cell lines (REH, KOPN41) and found that at least three chromosomal imbalances were involved in all patient samples and cell lines. Recurrent regions of chromosomal imbalances (found in > 10% of clinical samples) were gain of chromosomes 10 (17 %) and 21q (25 %) and loss of chromosomes 2p11 (100 %), 12p13.2 (87 %), 9p21.3 (29 %), 9p13.2 (25 %), 12q21.3, (25 %), 3p21 (21 %), 6q21 (17 %), 4q31.23 (17 %), 11q22-q23 (13 %) and 19q13.11-q13.12 (13 %). The two cell lines showed gain of 21q22.12-qter and loss of 2p11.2, 9p21.3, 12p13.2, and 12q21.3. Among these, six regions of loss (2p11, 3p21, 4q31.23, 9p13.2, 12q21.3 and 19q13.12) have not been identified previously by conventional CGH in TEL-AML1 leukemia. Representative genes involved in the regions of loss were Igkappa (2p11), TEL (12p13.2), p16INK4a/ARF (9p21.3), Pax5 (9p13.2), BTG1 (12q21.3), LIMD1 (3p21), AIM1 and BLIMP1 (6q21), NR3C2 (4q31.23), ATM (11q22-q23), and PDCD5 (19q13.11-q13.12), while the region of gain at 21q contained RUNX1. These findings suggest that, in addition to TEL previously reported as deleted, genes involved in cell cycle regulation, p53 pathways and apoptosis are also often deleted. Our array-CGH obtained data may provide further insights into the molecular basis for the development of TEL-AML1 leukemia.

Amplification of 6p21 has been detected in various solid tumor and hematological neoplasms. These include hepatoma, osteosarcoma, pancreatic cancer, bladder cancer, and mantle cell lymphoma . However, no candidate target genes in this amplified region have been identified. In diffuse large B-cell lymphoma (DLBCL), no amplification has been reported. Recently, we established genome-wide array comparative genomic hybridization (array CGH) consisting of 2300 BAC clones that can survey a whole genome at the density of an average size of 1.3 Mb. The array CGH detected amplification at 6p21 in 12 of 70 (24%) DLBCL patients (Tagawa et al., Cancer Res. in press) and one DLBCL cell line, SUDHL-9. We next made high density array glass for 6p21 region with 23 BAC clones covering 3 Mb. We found that the minimal common region of amplification was approximately 2Mb in size. In this region, there are 27 known genes including cyclin D3. The cyclin D3 has been reported to be overexpressed in a case of DLBCL with t(6;14)(p21.1;q32.3). We have analyzed expression level of those genes in the minimal common region of amplification, and found that five genes, BYSL, cyclin D3, TBN, KIAA0240, and TBCC were overexpressed. These results indicate that not only cyclin D3 but also other genes are also possible target genes for the 6p21 amplification.

Mantle cell lymphoma (MCL) is characterized by 11q13 chromosomal translocation and CCND1 overexpression, but the additional genomic changes are also important for lymphomagenesis. To identify the genomic aberrations of MCL in higher resolution, we analyzed 29 patient samples and seven cell lines (SP-53, Z-138C, Granta 519, REC-1, NCEB-1, Jeko-1 and JVM2) using array-based comparative genomic hybridization (array CGH) that consisted of 2,304 BAC/PAC artificial chromosome clones, covering the whole genome at a 1.3 mega base resolution. Tumor specimens obtained from 29 MCL patients, comprising 16 males and 13 females all from the Aichi Cancer Center, were included in the study. The median age of the patient was 67 years (49–92 years old). 67% of cases were leukemic, 93% were in an advanced stage III-IV, 30% had elevated LDH, 18% had a poor performance status and 78% had more than one extranodal site of involvement. Genomic profiles of MCL were significantly different from those of other B-cell lymphomas such as diffuse large B-cell lymphoma (n=66) (Cancer Res 2004: 64, in press) and follicular cell lymphoma (n=13). Frequent imbalances detected by array CGH in patients were gains of chromosomes 3q26, 7p21, 6p21, 8q13-8q24, 10p12 and 17q23 (frequencies: 21%–48%), and losses of chromosomes 1p36, 1p21–p32, 1q42, 2p11, 2q13, 5q21, 6q16–q24, 8p12–p23, 9p21–p24, 10p14, 9q22, 11q22, 13q21, 13q34, 17p11.2–p13, 19p13 and 22q12 (frequencies: 17%–83%). Among these gains and losses, patents with a gain of 8q13-q24, and losses of 1p22–p32, 6q16–q24, 8p12–p23 and 17p11.2–p13 showed prognostically inferior survival rate to those without such a gain or losses (P

Diffuse large B-cell lymphoma (DLBCL) is the most common type of non-Hodgkin’s lymphoma (NHL), accounting for 30 – 40% of adult NHL. Genomic alterations in DLBCL have been investigated by various methods and many regions of amplifications and losses have been detected. Genomic deletions of the 3p arm have been reported in many solid tumors and leukemias, but no study published to date has reported genomic deletions of this region in DLBCL. Recently we demonstrated that 3p14.2 was deleted in approximately 30% of DLBCL patients and cell lines by use of a genome-wide array-comparative genomic hybridization (array-CGH) (Tagawa et al., 2004). For a more detailed examination of the genomic losses at 3p14.2, here we made use of contig BAC array for 3p14.2, and found that 12 of 27 DLBCL samples displayed losses. All of the deleted regions were located within the Fragile Histidine Triad (FHIT) gene, and the most frequently region of loss was mapped to 0.4 Mbp of the region encompassing the introns 4 and 5 and exon 5 of the FHIT gene. Genomic deletions of the FHIT gene have been observed in most common forms of cancer and it is therefore conceivable that the FHIT gene is the target one of the genomic deletion at 3p in DLBCL. To investigate the relationship between genomic deletions and transcript alterations, we performed nested reverse transcriptase-polymerase chain reaction (RT-PCR) analysis. Aberrant transcripts or loss of expression was detected in 33% (19 of 57) of the DLBCL samples, and the lost exons of the aberrant transcripts were correlated with genomic deletions detected by array CGH. We also investigated the CpG island methylation status of the promotor of the FHIT gene by means of methylation specific PCR (MSP). One case with genomic loss of the FHIT gene, and three cases without genomic loss showed methylated allele. These findings indicate that 1) Loss of genomic material at 3q14.2 is responsible for exon losses of the FHIT gene, and 2) Genomic loss of the FHIT gene is one of the causes of the generation of aberrant transcripts. 3) Both genomic deletions and methylations are involved in the causes of inactivation of the FHIT gene.