ALBERT

Description: 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.

Description: BACKGROUND Bone marrow examination is essential in diagnosis, staging and monitoring of various hematologic disorders. The aspirate smears and core biopsy are complementary samples; current clinical benchmarks recommend an optimal core sample length of at least 15-20 mm. We assessed the core length in 2 cross-sectional cohorts from 2001 and 2011 in 32 academic medical centers from the US and Canada, the first such study to date. METHODS After IRB approval, participants collected data from pathology reports (including the preprocessing length) and measured aggregate postprocessing and evaluable marrow length using a uniform validated methodology on 100 consecutive marrow samples in 2001 and 2011 at each institution. Deidentified data was centralized at Roswell Park Cancer Institute (RPCI) and centers were anonymized. A total of 6374 samples were accepted for statistical analysis, performed using SAS (v. 9.4 or higher; SAS Institute, Cary, NC) at RPCI. Relationship between core length and NCCN status, geographic location, gender, age, and staging was assessed using the PROC MIXED and PROC GLIMMIX procedure using a random center effect and a nominal significance level of 0.05. RESULTS The study cohort included 56% men and 44% women, mean age 51 (range, 1-102) years, 88% adults (³18) and 12% children (

Description: A20, a negative regulator of NF-κB, has been implicated as a tumor suppressor gene in multiple types of B-cell lymphoma. AIDS-related lymphomas (ARLs) are high-grade B-cell lymphomas that are frequently associated with EBV infection. We examined a panel of ARLs for A20 alterations. FISH showed A20 deletion in 6 of 33 cases (18%). A20 mutations were found in 3 of 19 cases (16%), including 2 cases with deletions of the comple-mentary allele. Immunohistochemistry showed the absence of A20 protein in 7 of 55 samples (13%). In contrast to reports in Hodgkin lymphoma in which EBV infection and A20 alteration are mutually exclusive, A20 inactivation was observed in both EBV+ and EBV− cases. The EBV latent membrane protein 1, which activates NF-κB, was not expressed in 12 of 13 cases with A20 loss. In ARLs loss of A20 may be an alternative mechanism of NF-κB activation in the absence of latent membrane protein 1 expression.

Description: Abstract 802 Introduction: AIDS related lymphomas (ARL) are a heterogeneous group of lymphoproliferative disorders that are frequently associated with Epstein Barr virus (EBV) infection. EBV expresses latent viral oncoproteins that constitutively activate the transcription factor NF-κB, a potent inducer of genes involved in B cell survival and proliferation (Keller SA et al, Blood 2006). Lymphomas that are not associated with EBV can also display increased NF-κB activity and recent reports have described mutations in regulators of NF-κB in subsets of B cell lymphomas. One of the frequently mutated regulatory genes is TNFAIP3, which encodes A20, an ubiquitin modifying enzyme involved in the termination of NF-κB signaling. Mutations resulting in the inactivation of A20 have been found in a significant proportion of marginal zone lymphoma (Novak U et al, Blood 2009), classical Hodgkin lymphoma, primary mediastinal B cell lymphoma (Schmitz R et al, J Exp Med 2009), and diffuse large B cell lymphoma (Compagno M et al, Nature 2009). In ARL the incidence of alterations in A20 and the relationship with EBV infection has not been described. Materials and Methods: We evaluated archival formalin fixed paraffin embedded tissue samples of ARL for genetic alterations in A20. Tissue was collected through an international collaboration between Weill Cornell Medical College in New York, USA and Siena University in Siena, Italy. A tissue microarray with 46 cases of ARL was prepared and characterization of lymphoma subtype and EBV viral status were determined by immunohistochemistry and in situ hybridization for Epstein-Barr encoded RNA. Fluorescent in situ hybridization (FISH) was used to evaluate for genomic deletions in A20, and translocations of cMYC, BCL-2 and BCL-6. Direct sequencing of the coding region and splice sites of A20 was performed to evaluate for additional genetic alterations. Immunohistochemistry was used to evaluate for the presence of A20 protein. Results: Fluorescent in situ hybridization revealed A20 monoallelic or biallelic deletion in 6 of 25 cases (24%). A20 point mutations were found in 3 of 23 cases (13%). Nonsense mutations coding for a premature stop codon in exon 2 were seen in 2 cases. The third case was found to have a missense mutation in exon 7 resulting in an amino acid change. Two of the 3 cases with an A20 point mutation had A20 deletion in the complementary allele indicating biallelic alteration of the A20 gene. Immunohistochemistry for A20 was performed and is reported for the first time in this abstract. Absence of A20 protein was demonstrated in 4 of 33 samples (12%). Included among the cases negative for A20 on immunohistochemistry is the single case with biallelic A20 deletion demonstrated by FISH. In total 10 of 39 (26%) cases with adequate sample for evaluation were determined to have inactivation of A20 by FISH, sequencing, immunohistochemistry, or a combination. A20 inactivation was seen among all histologic subtypes of ARL including Burkitt lymphoma (n=2), diffuse large B cell lymphoma of the germinal center B cell (n=2) and non-germinal center B cell (n=2), plasmablastic lymphoma (n=3) and B cell lymphoma, unclassifiable, intermediate between BL and diffuse large B cell lymphoma (n=1). Interestingly, the incidence of EBV infection was higher in cases with A20 inactivation than in those with intact A20. EBV was present in 6/10 cases with A20 alteration (60%) vs. 8/29 cases with intact A20 (28%). The EBV latent viral protein LMP-1, which activates NF-κB, was not expressed in cases with A20 alteration. Conclusions: This is the first report to demonstrate A20 inactivation in EBV-associated lymphoma. A20 molecular analysis has been previously reported in Hodgkin Lymphoma (HL) where A20 inactivation and EBV infection were found to be almost mutually exclusive (Schmitz R et al, J Exp Med 2009). The EBV gene expression pattern differs in HL and ARL. In HL EBV expresses the viral oncoprotein LMP-1, which leads to constitutive activation of NF-κB. In ARL viral gene expression is more heterogeneous and in this cohort of ARL, LMP-1 was not expressed in any of the cases with EBV infection and A20 loss. Our data indicate that A20 may represent a tumor suppressor gene in a significant subset of ARL and that A20 inactivation may be associated with positive EBV status. In EBV related lymphoma inactivation of A20 may be an alternative mechanism of NF-κB upregulation in the absence of LMP-1. Disclosures: No relevant conflicts of interest to declare.