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Introduction Blastic plasmacytoid dendritic cell neoplasm (BPDCN) is a rare and aggressive hematological malignancy with multi-organ and frequent skin involvement, and poor clinical outcomes. Based on the limited available data, the estimated incidence is 0.44% of all hematologic malignancies, representing less than 1% of acute leukemias, and 0.7% of cutaneous lymphomas. Due to the rarity of this entity, there have been relatively few studies characterizing the molecular profile of BPDCN. We examined a cohort of 51 patients with BPDCN using OncoScan chromosome microarray, which provides genome-wide copy number abnormality (CNA) analysis. Methods An international cohort of BPDCN cases were collected from centers in Brazil (Laboratorio de Patologia, Botucatu), Swtizerland (University of Zurich), France (Hospital St. Louis, Paris), Peru (Instituto Nacional de Enfermedades Neoplasicas, Lima), Canada (Department of Pathology, University of Montreal), Italy (Derpartment of Pathology, University of Bologna), and US (Department of Pathology - The Ohio State University, Department of Hematopathology - MD Anderson Cancer Center; and Department of Pathology - University of Virginia). A total of 58 tissue blocks from 51 patient samples were retrieved. The diagnosis of BPDCN was done and confirmed by at least three independent hematopathologists or dermatopathologists in accordance with the WHO classification (Lyon 2017). For the purpose of the molecular analysis substratification, cases were classified as 'BPDCN' if they were positive for TCF4, and 'BPDCN-like' if they were negative for TCF4. Immunohistochemistry for CD123, CD4, and CD56 was performed in all cases. Exclusion criteria included expression of MPO, lysozyme, CD3, CD19, CD20, CD22, and/or EBV. DNA was extracted from FFPE samples via standard techniques and processed on OncoScan CNV Plus microarray (ThermoFisher Scientific) according to manufacturer's recommended protocol. Copy number abnormalities and select single nucleotide variants and insertions/deletions (74 mutations in 9 genes) were analyzed on Chromosome Analysis Suite software (ChAS v4.1; ThermoFisher Scientific). Additional analysis was performed using Nexus Copy Number (BioDiscovery, version 10.0). Results To date, we have successfully analyzed 45 cases of BPDCN with Oncoscan, revealing widespread CNA in the vast majority of cases (44/45; 98%). Alterations of chromosome 9 were common in this cohort, particularly CNAs involving CDKN2A/B at 9p21.3. Twenty-five cases (56%) demonstrated CNA including CDKN2A/B, with ten of these cases demonstrating a homozygous loss of CDKN2A/B (22%). Alterations of chromosome 13 were also frequently detected with loss of RB1 (located at 13q14.2) detected in 24 cases (53%). The RUNX1 gene (21q22.12) was a common target of CNAs in this cohort, seen in nine cases (20%). Eight of these cases showed a copy number gain of RUNX1, which is a recurrent finding in a variety of hematological malignancies, particularly myeloid neoplasms. The remaining case with RUNX1 CNA showed a focal, homozygous loss of the gene, demonstrating that dysregulation of RUNX1 through CNA is a common event in BPDCN. We observed frequent deletions of ETV6 (53%), IKZF1 (33%), and TP53(16%) in our cohort. The ARHGAP26 gene (5q31.3), which is associated primarily with juvenile myelomonocytic leukemia, was included in CNA in 13 cases (29%), with both gains and losses observed in this cohort. Oncoscan can detect a limited number of single nucleotide variants in nine genes that are frequently mutated in cancers (BRAF, EGFR, IDH1, IDH2, KRAS, NRAS, PIK3CA, PTEN, and TP53). Mutations were detected in ten cases (22%), with NRAS and TP53 variants detected in three cases each and KRAS and IDH2 variants detected in two cases each. Conclusions Our preliminary data demonstrates complex genomic alterations in BPDCN, with the RB1 locus on chromosome 13, the CDKN2A/B locus on chromosome 9, and the ETV6 locus on chromosome 12 most commonly detected. However, widespread genomic alterations were detected involving a variety of cancer-associated genes further characterizing CNA in BPDCN. Analysis of additional BPDCN cases is progress. Disclosures Khoury: Kiromic: Research Funding; Angle: Research Funding; Stemline Therapeutics: Research Funding. Porcu: Viracta: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Innate Pharma: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; BeiGene: Membership on an entity's Board of Directors or advisory committees, Research Funding; Incyte: Research Funding; Daiichi: Honoraria, Research Funding; Kiowa: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Spectrum: Consultancy; DrenBio: Consultancy. Gru: StemLine: Honoraria, Research Funding, Speakers Bureau; CRISPT Therapeutics: Research Funding; Innate Pharma: Research Funding.

LMO2 regulates gene expression by facilitating the formation of multipartite DNA-binding complexes. In B cells, LMO2 is specifically up-regulated in the germinal center (GC) and is expressed in GC-derived non-Hodgkin lymphomas. LMO2 is one of the most powerful prognostic indicators in diffuse large B-cell (DLBCL) patients. However, its function in GC B cells and DLBCL is currently unknown. In this study, we characterized the LMO2 transcriptome and transcriptional complex in DLBCL cells. LMO2 regulates genes implicated in kinetochore function, chromosome assembly, and mitosis. Overexpression of LMO2 in DLBCL cell lines results in centrosome amplification. In DLBCL, the LMO2 complex contains some of the traditional partners, such as LDB1, E2A, HEB, Lyl1, ETO2, and SP1, but not TAL1 or GATA proteins. Furthermore, we identified novel LMO2 interacting partners: ELK1, nuclear factor of activated T-cells (NFATc1), and lymphoid enhancer-binding factor1 (LEF1) proteins. Reporter assays revealed that LMO2 increases transcriptional activity of NFATc1 and decreases transcriptional activity of LEF1 proteins. Overall, our studies identified a novel LMO2 transcriptome and interactome in DLBCL and provides a platform for future elucidation of LMO2 function in GC B cells and DLBCL pathogenesis. © 2012 by The American Society of Hematology., E.C. is partially supported by the Fundacion Alfonso Martin Escudero. I.S.L. is supported by National Institutes of Health (NIH) grants CA109335, CA122105, and U56 CA112973, and the Dwoskin Family and Recio Family foundations. I.S.G. is supported by MICINN (SAF2009-08 803), and by MEC OncoBIO Consolider- Ingenio 2010 (Ref. CSD2007-0017). All Spanish funding is cosponsored by the European Union FEDER program.

We examined the types of Epstein-Barr virus–associated nuclear antigen-1 (EBNA-1) gene carboxy (C)-terminal mutations occurring in Hodgkin’s disease (HD) and reactive tissues from two different geographic regions. Previously reported EBNA-1 C-terminal region amino acid sequence variants, based on the amino acid at codon 487, include Prototype (P)-ala, which is found in the B95.8-derived prototype virus, P-thr, Variant (V)-leu, V-val, and V-pro. Using polymerase chain reaction (PCR) to amplify portions of the EBNA-1 gene, followed by DNA sequencing, we found a single EBNA-1 gene sequence variant in each tissue, whether reactive or neoplastic and whether from Brazil or the United States. Variant EBNA-1 gene sequences were more common in both neoplastic and non-neoplastic tissues from different geographic areas than the so-called prototype sequence. In the 17 Brazilian HD cases, 4 cases had P-thr variants and 13 had V-leu variants. In the six reactive tissues from Brazil, one had a P-ala variant, two had P-thr variants, and three had V-leu variants. In the 12 American HD cases, 2 had P-ala variants, 6 had P-thr variants, and 4 had V-leu variants. The 11 American reactive tissues included 2 P-ala variants, 5 P-thr variants, and 4 V-leu variants. In both countries, there were similar variant EBNA-1 sequences present in normal tissues and HD cases. Compared with the P-ala and P-thr cases, the V-leu cases were more likely to have the 30-bp latent membrane protein 1 (LMP1) gene deletion (P = 0.0075). In addition, cases of HD with the V-leu were statistically associated with a substitution of asparagine for glutamine at codon 322 of the C-terminal portion of the LMP1 gene. Our results suggest that any variation in EBNA-1 gene sequence is caused by a polymorphism present in pre-existing viral strains in the underlying population, and not a mutation occurring during oncogenesis.

Abstract 3471 PRDM1/Blimp1, a lymphoma tumor suppressor and master regulator of plasma cell differentiation, is inactivated by genomic mutation and deletion in a subset of activated B-cell-type diffuse large B-cell lymphomas (DLBCL), underscoring the importance of terminal differentiation impairment in lymphoma pathogenesis. The role of PRDM1 inactivation in Burkitt lymphoma (BL) is currently not known. Two findings, however, may suggest possible PRDM1 involvement in BL pathogenesis. First, although PRDM1 protein is consistently absent in BL, about 40% of BL cases are positive for IRF4/MUM1, suggesting asynchronous expression of these two proteins. Secondly, it has been hypothesized that microRNA-127 overexpression in EBV+ BL may promote BL development through down-regulation of PRDM1. Sequence analysis of the coding region of PRDM1 in BL cell lines and primary tumors did not demonstrate any mutational changes. To investigate whether epigenetic inactivation of PRDM1 may play a pathogenetic role in BL, BL cell lines and primary cases were bisulfite sequenced to assess the methylation status of 41 CG dinucleotides in a 601 base-pair region spanning the distal promoter (DP) and a CG island that extends from the proximal promoter to the first exon of PRDM1. These include (i) 11 BL cell lines, 9 EBV+ (4 latency I, 2 Wp-restricted, and 3 with latency III drift) and 2 EBV-; (ii) 7 EBV+ lymphoblastoid cell line (LCL); (iii) 62 primary BL cases, formalin-fixed, paraffin-embedded tissues (FFPE); (iv) 4 B cell lymphoma, unclassifiable, with features intermediate between DLBCL and BL (BCL-U), FFPE. Naïve, germinal center (GC) and memory B cells sorted by flow cytometry were also analyzed as controls. Hypermethylation in PRDM1 promoter and exon 1 was seen in all EBV+ BL cell lines except one with latency III, but not in EBV- BL lines. None of the LCL demonstrated PRDM1 hypermethylation, implying that EBV is unlikely to mediate PRDM1 hypermethylation directly. Methylation status of PRDM1 was successfully determined in 39 BLs (30 sporadic, 9 HIV-related) and 5 BCL-Us (4 sporadic, 1 HIV-related). Hypermethylation was seen in 11 BLs and 1 BCL-U (28.6% of total). All the methylated cases were EBV(+) (p=0.004). Overall, 12 of 28 (43%) EBV(+) BL and BCL-U cases exhibited hypermethylation. PRDM1 hypermethaylation was independent of BL subtype and MUM1/IRF4 expression status. To determine if PRDM1 hypermethylation can potentially repress PRDM1 transcription, BL cell lines Ramos (PRDM1 unmethylated) and Mutu1 (PRDM1 hypermethylated) are treated with IL-21 (50ng/mL), a cytokine that can mediate terminal differentiation by inducing PRDM1, for 2 to 5 days. While PRDM1 is induced by IL-21 in Ramos, levels of PRDM1 are not significantly increased in Mutu1, even though STAT3, a downstream mediator of IL-21, is activated in the latter. This finding suggests that PRDM1 hypermethylation has the potential to repress PRDM1 transcription in the presence of PRDM1-inducing signals. This function, however, may be pathogenetically more relevant in a BL precursor cell than in the final BL tumor cell. EBV+ BL is thought to derive from an EBV-infected late germinal center (GC) B cell, which harbors c-myc translocation and begins, though abortively, memory B cell differentiation and adopts an EBV latency I form. BL consistently express BCL6, a known PRDM1 transcription repressor, and harbor very low levels of PRDM1 mRNA. This suggests that high BCL6 expression, rather than PRDM1 hypermethylation, may be the primary determinant of its repressed transcription seen in BL. Indeed, PRDM1 mRNA expression is 25 to 1200 fold higher in LCLs which harbor very low levels of BCL. Furthermore, reducing expression of MTA3, which forms a complex with BCL6 and mediates PRDM1 transcription repression, in Daudi increases PRDM1 transcript levels. Therefore, we hypothesize that PRDM1 hypermethylation in BL more likely represents a memory of an epigenetic event that functions in the earlier stage of BL pathogenesis. PRDM1 hypermethylation may function to repress its transcription in a BL precursor cell as it is exposed to inducing signals, e.g. IL-21, during GC transit. Our study expands the spectrum of B cell lymphomas in which PRDM1 plays a tumor suppressor role, and supports the importance of impairment of terminal differentiation in the pathogenesis of a subset of aggressive B cell lymphomas. Disclosures: No relevant conflicts of interest to declare.

Abstract 4858 Background The proportion of classical Hodgkin lymphoma that is associated with Epstein - Barr virus (EBV+ cHL) varies between geographic locations, ethnic groups and socio-economic status. In Western Europe, approximately 30% of cHL is EBV associated, while studies from Brazil have shown a 40–100% incidence of EBV+ cHL. We previously demonstrated that specific HLA-A alleles are associated with EBV+ cHL in the Western European population, with HLA-A1 being a risk allele and HLA-A2 being protective. We also demonstrated that in the northern Chinese population HLA-A*02:07 is a protective allele for EBV- and a risk allele for EBV+ cHL. In both China and Western Europe, EBV involvement is associated with a retained expression of HLA class I by the Hodgkin Reed-Sternberg (HRS) cells. The current study focuses on EBV, HLA class I expression and associations with HLA-A alleles in a group of Brazilian cHL patients. Patients and Methods A total of 158 cHL consultation cases (2005-2007), obtained throughout Brazil were available at the Consultoria em Patologia, Botucatu-São Paulo, Brazil. Data on sex and age were retrieved for all cases. Hematoxilin & Eosin stained tissue sections were used to reclassify the histological subtypes according to the WHO classification. EBV status was determined by EBER in-situ hybridization (ISH) using tissue micro arrays with clearly identifiable tumor cells. Membranous expression of HLA class I was determined by immunohistochemistry using HC10 (class-I heavy chain) and beta-2-microglobulin monoclonal antibodies. HLA-A1 and HLA-A2 qPCR was performed on DNA isolated from tissue sections. The qPCR was validated using known HLA-genotype samples to discriminate between non carriers and heterozygous or homozygous carriers for HLA-A1 and HLA-A2. Control data on HLA typing was available for 46,044 blood bank donors from Brazil. Results The median age of the patients was 25 years (3-83) with a male to female ratio of 1:2. The nodular Sclerosis (NS) histopathologic subtype was the most frequent (78%) and Mixed Cellularity (MC) was observed in 18% of the patients. Tumor cells in 34% of the patients were EBV positive (MC 79% and NS 25%). The age incidence was bimodal with a first prominent incidence peak between 11 and 30 years and a second peak in the 61–70 years age group. The first incidence peak was not associated with EBV (22% EBV+), while the second peak was (58% EBV+). In the pediatric age group (10 yr or less), 73% of the patients were EBV+. Membranous expression of HLA class I was significantly more often retained in HRS cells of EBV+ cHL compared to EBV- cHL, 53% vs. 17% (p=0.0004). The HLA-A1 carrier frequency was significantly increased in EBV+ cHL (32%) compared to controls (18%, p=0.02). In contrast, the HLA-A2 carrier frequency in EBV+ cHL was significantly reduced compared to controls, 28% vs. 45% (p=0.02). Conclusions We observed a bimodal age incidence distribution with a prominent early peak in the second to third decade. EBV positivity was especially high in young children and MC subtype and was associated with HLA class I expression by the HRS cells. The genetic risk pattern was similar to the Western European population, with HLA-A1 as a risk and HLA-A2 as a protective allele for developing EBV+ cHL. Disclosures: No relevant conflicts of interest to declare.