ALBERT

Description: The contamination of aquatic environments by microplastics has been largely documented in the last years, especially in oceans, rivers, and lakes, but their occurrence in remote mountain lakes has been scarcely considered. This work aims to investigate the presence and abundance of microplastics and fibres in a remote, alpine, and uninhabited lake in Switzerland (Sassolo). In this study, the water column as well as the sediments were analysed. The isolation of microplastics and fibres from the samples of the sediment was achieved with a digestion process using H2O2 and a density separation technique with NaI. Classification of microparticles (from 5 mm to 125 μm) was first developed with an optical microscope. Infrared spectroscopy was then used to identify and characterize the chemical nature of the microplastics and fibres. On average, 2.6 microplastics and 4.4 fibres per litre were identified in the water column. On the other hand, the results of the sediment samples revealed significant fibre concentrations compared to plastic microparticles (514 fibres and 33 microplastics per kilogram). The most abundant types of microplastic identified in the samples were composed of polyethylene (PE) and polypropylene (PP). Microplastic and fibre sources were not determined, but it is likely that the number of human activities in this area as well as aerial deposition are contributing to contaminate this remote environment with microplastics and fibres.

Description: Background: Diffuse large B-cell lymphoma (DLBCL) is clinically and biologically heterogeneous. Whereas most patients are cured following conventional therapy with rituximab, cyclophosphamide, doxorubicin, vincristine and prednisone (R-CHOP), a substantial minority are refractory or relapse. Identifying patients at high risk of poor outcomes at the time of diagnosis will make it possible to better choose alternative or experimental therapies. TCF3 (also called E2A) is a transcription factor required in B-lymphopoiesis. Gain-of-function somatic mutations that alter TCF3 or its regulatory partners are associated withBurkitt lymphoma (BL) but not DLBCL. These published findings suggest that biologically-defined, clinically-important subtypes of mature B-cell-derived lymphomas may be distinguished from one another based on differential expression of genes regulated by a master regulator of B-lymphoid biology, namely TCF3. Therefore, in the current study, we hypothesized that biologically- and clinically-important subtypes of DLBCL are distinguishable based upon differential expression of genes that are regulated in B-cells by TCF3. Methods: Fifty-nine subjects who presented with de novo DLBCL at Kingston General Hospital from 2001 to 2010 were identified for inclusion based on the availability of clinical data and sufficient biopsy material for extraction of adequate RNA. We used publically-available microarray-based gene expression and clinical data to identify 99 candidate TCF3 target genes whose differential expression was associated with 3-year overall survival following R-CHOP therapy; these transcripts were represented in aNanoStringCodeSet. Total RNA was purified from cores harvested from representative areas of diagnostic, pre-treatment, formaldehyde-fixed, paraffin-embedded (FFPE) biopsy samples. Twenty-five transcripts whose abundance, individually, was associated with 3-year overall survival in our own cohort were chosen for further study. Results: Unsupervised hierarchical cluster analysis based on relative expression of the 25 TCF3 target genes resolved all except two cases into distinct clusters, denoted A and B, that contained 21 and 36 cases, respectively (heat map at left). In comparing these clusters, Cluster B was associated with rearrangement of MYC (p=0.02) as determined by FISH; no statistically significant associations were evident with age, sex, IPI score, B-symptoms, bulky disease or expression of MYC or BCL2 as determined by immunohistochemistry. Kaplan Meyer (KM) analysis revealed strikingly inferior overall and event-free survival (p=0.0005 for each) for Cluster A cases (KM curve at right). Conclusions: Our findings demonstrate that differential expression of TCF3 target genes ascertained in pre-treatment FFPE biopsy samples of de novo DLBCL defines biologically distinctive subsets of cases with distinctive clinical outcomes following R-CHOP therapy. If validated in an independent cohort, these results may inform both clinical management and the development of new targeted therapies for high-risk cases. Figure 1 Figure 1. Figure 2 Figure 2. Disclosures No relevant conflicts of interest to declare.

Description: Abstract 1586 Background Mantle cell lymphoma (MCL) is a subtype of B-cell non-Hodgkin lymphoma (NHL) characterized by the t(11;14) translocation and concomitant over-expression of cyclin D1. MCL has a variable natural history; while some patients have prolonged survival similar to other indolent B-cell lymphomas, most follow an aggressive course with short survival. While the t(11;14) is pathognomonic of MCL, it is not necessary for disease pathogenesis, as a subset of MCL cases lack the translocation. Furthermore, in vivo models demonstrate that cyclin D1 over-expression alone is unable to bring about the disease, and that deregulation of additional cellular pathways is required for its pathogenesis. Assessment of microRNA (miR) expression in MCL may help determine mechanisms of gene deregulation and reveal pathways involved in disease pathogenesis. In this study we examined MCL in relation to both aggressive and indolent B-cell NHL to determine a miR signature that characterizes MCL. Design and Methods Total RNA from a training set of 36 B-cell NHL cases (19 indolent and 17 aggressive) and 32 MCL was applied to a high-throughput quantitative real-time PCR platform assessing the expression of 365 miRs [TaqMan Human MicroRNA Array v1.0 (Early Access) or TLDA]. miRs that were differentially expressed between MCL and aggressive NHL, and between MCL and indolent NHL were then validated using RNA from a second, independent, set of B-cell NHL cases (28 indolent and 28 aggressive) and 50 MCL cases. Validated miRs were determined and potential targets for each miR were examined. A map of targets common to the MCL miR signature was created, revealing important proteins involved in MCL pathogenesis. Results 66 miRs (11 over-expressed, 55 under-expressed) were differentially expressed between MCL and aggressive B-cell NHL and 8 miRs (7 over-expressed, 1 under-expressed) were differentially expressed between MCL and indolent B-cell NHL (false discovery rate = 0.2). 6 miRs from each group were chosen for validation in an independent set of MCL and NHL cases. Of these 12 miRs, 7 miRs validated (2 were under-expressed in MCL relative to aggressive B-cell NHL, and 5 were over-expressed in MCL relative to indolent B-cell NHL). Genes and pathways involved in disease pathogenesis are most likely targeted by multiple miRs. We thus determined a set of 123 genes predicted to be targets of this MCL miR signature, based on five miR target prediction databases from the mirDIP (microRNA data integration) portal. These genes were significantly enriched for focal adhesion and integrin signalling, proteasome-mediated degradation, and the PI3K signalling pathway. Conclusions Using the largest set of MCL cases evaluated to date, a 7-miR signature characteristic of MCL was discovered. The gene targets of these miRs are enriched for roles in proteasome-mediated protein degradation, consistent with the reported sensitivity of MCL to proteasome inhibitors. In addition, these miRs are predicted to be involved in regulation of PI3K/AKT signalling, confirming reports of the importance of this pathway in MCL pathogenesis. Enrichment of target genes involved in focal adhesion and integrin signalling indicate the importance of MCL-stromal interactions and motivates further study into the role of the tumor microenvironment in MCL pathogenesis. Disclosures: No relevant conflicts of interest to declare.

Description: The E2A locus encodes transcription factors, called E12 and E47, involved in lineage-specific cellular differentiation. The locus is also involved in chromosomal translocations associated with acute lymphoblastic leukemia, the most common of which results in expression of the oncoprotein E2A-PBX1. We showed recently that direct interaction between E2A and the histone acetyl-transferase (AT) and transcriptional co-activator proteins CBP/p300 is required in leukemogenesis by E2A-PBX1. E2A proteins have also been shown to interact with another AT/co-activator, p/CAF. Interaction with these AT proteins results in acetylation of E2A proteins themselves. Here we map the acetylated lysine residues within the oncogenic portion of E2A proteins and begin to elucidate some functional correlates of E2A acetylation. Our results indicate that the isolated AT domain of p/CAF as well as full-length p/CAF were capable of acetylating E2A. Interestingly, full-length p300 was capable of acetylating E2A while an isolated AT domain was unable to acetylate E2A, suggesting that additional domains of CBP/p300 are required to mediate E2A acetylation. We demonstrate that both p300 and p/CAF can interact directly with E2A, independent of the known interaction between p300 and p/CAF. These co-activators do, however, appear to co-operate to achieve maximal E2A acetylation. Mutagenesis-based mapping studies indicate that several lysines are substrates for acetylation. Of particular interest, a conserved lysine residue (K34) located within the N-terminal transcriptional activation domain (AD1) is acetylated in vitro by p/CAF. K34 is located within a GKXXP consensus sequence, suggested to be a recognition motif for acetylation by several AT enzymes including CBP/p300 and p/CAF. Conservative replacement of K34 with arginine (i.e., K43R) substantially impairs transcriptional activation of a luciferase reporter gene by E2A, suggesting that post-translational modification of this residue may play an important functional role. Consistent with a role for acetylation, relative to some other lysine-dependent modification, we were unable to demonstrate sumoylation or ubiquitination of the N-terminus of E2A. Therefore, we have found that acetylation by AT/co-activator proteins contributes to transcriptional regulation by the functionally critical N-terminal activation domain (AD1) of E2A proteins. The mechanisms by which this acetylation event is regulated and how it contributes to target gene induction by E2A are not clear. It seems plausible that the acetylation status of AD1 could be determined by upstream signaling events and acetylation of E2A could modulate interactions with transcriptional co-regulators, DNA or chromatin. Further studies to investigate these possibilities are underway. In particular, results using an amino acid substitution that mimics acetylation of AD1 (K34Q) will be presented.

Description: E-proteins are critical transcription factors in B-cell lymphopoiesis. E2A, 1 of 3 E-protein–encoding genes, is implicated in the induction of acute lymphoblastic leukemia through its involvement in the chromosomal translocation 1;19 and consequent expression of the E2A-PBX1 oncoprotein. An interaction involving a region within the N-terminal transcriptional activation domain of E2A-PBX1, termed the PCET motif, which has previously been implicated in E-protein silencing, and the KIX domain of the transcriptional coactivator CBP/p300, critical for leukemogenesis. However, the structural details of this interaction remain unknown. Here we report the structure of a 1:1 complex between PCET motif peptide and the KIX domain. Residues throughout the helical PCET motif that contact the KIX domain are important for both binding KIX and bone marrow immortalization by E2A-PBX1. These results provide molecular insights into E-protein–driven differentiation of B-cells and the mechanism of E-protein silencing, and reveal the PCET/KIX interaction as a therapeutic target for E2A-PBX1–induced leukemia.

Description: Abstract 4630 Persistence of a mass after first-line treatment is a common problem in nodular sclerosis Hodgkin lymphoma (NSHL). Up to 64% of patients demonstrate residual abnormalities on computed tomography (CT) after therapy, but only 42% of those patients will relapse on follow-up. This is primarily caused by the inability of CT to distinguish viable tumor tissue from fibrosis. Clinicians are faced with the dilemma of whether to pursue second-line treatment for a mass that may be simply scar tissue. The ability to predict which patients are at higher risk for residual mass following curative treatment can aid in the planning of follow-up imaging modalities such as positron emission tomography (PET) scanning which can map out metabolically active tissue (i.e. tumor versus fibrosis) and the need for biopsy of a residual mass. This study was designed to test the hypothesis that the presence of abundant fibrosis in the initial biopsy predicts the presence of residual, post-therapy masses composed primarily of fibrotic tissue. Subjects were consecutive NSHL patients from the years 1996 to 2007 identified from our institution based on the availability of histology slides from the initial diagnostic biopsy, clinical follow-up data, and the results of post-treatment imaging investigations. The initial biopsies were reviewed by a lymphoma pathologist and resident without knowledge of the residual mass status. The proportion of the tissue consisting of fibrous material was graded as a percentage of the total biopsy tissue. The clinical charts were reviewed for baseline patient characteristics, cancer stage and the presence of a residual mass on CT scan 6 months after treatment. Of the 47 subjects included in the study, 25 had residual masses and 22 had none. Patients with increased fibrosis on initial biopsy were significantly more likely to have a residual mass after initial therapy (p=0.028). The degree of fibrosis was independent of gender, stage, and Hasenclever score. Degree of fibrosis was the only factor that was predictive of the presence of a residual mass. Of the 16 patients with residual masses with follow-up Gallium imaging, the result of the scan was more likely to be negative (indicating that the mass is not metabolically active) for patients with a high grade of initial fibrosis (p=0.148). Taken together, these results suggest that patients with increased fibrosis on their initial NSHL biopsy are more likely to have residual masses, but that these masses are less likely to be malignant. The results support the hypothesis that the degree of fibrosis at the initial NSHL biopsy is predictive of a post-treatment residual mass. These findings have potential implications for patient follow-up: clinicians whose patients have abundant fibrosis at initial biopsy may be reassured that a post-treatment residual mass is less likely to represent persistent malignancy and thus can be followed with functional imaging rather than pursuing unnecessary biopsies. Our results further reinforce the importance of functional imaging like PET, particularly in this patient population Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 800 Mantle cell lymphoma (MCL) is a B-cell non-Hodgkin lymphoma (NHL) accounting for ~6% of all NHL. It is sensitive to combination chemotherapy, but remission durations are short without approaches such as stem cell transplantation (SCT). Most patients are incurable, but the clinical course is variable, with some patients succumbing quickly, while others survive 〉10 years. MicroRNAs (miRs) are small, non-coding RNAs that regulate gene expression by inhibiting mRNA translation. miRs are useful in the prognostic assessment of tumors, but work to date examining differences between MCL and normal lymphoid tissues, have only identified 2 miRs involved in MCL prognosis (Zhao JJ, Blood, 2010; Di Lisio L, Leukemia, 2010). We used a novel approach to identify a prognostic miR signature in MCL. We hypothesized that a miR signature defining aggressiveness can be obtained by comparing miR expression profiles of aggressive NHL with indolent NHL, and that this signature when applied to a set of MCL cases, may aid in MCL prognosis. Total RNA was extracted from 135 formalin-fixed paraffin-embedded samples obtained at primary diagnosis (Table 1). RNA from a training set of 19 indolent and 20 aggressive NHL cases was analyzed on a high-throughput quantitative real-time PCR (qRT-PCR) platform assessing the expression of 365 miRs and 3 endogenous controls (TaqMan Human MicroRNA Array v1.0: TLDA, ABI) using the DDCt method. A two-sample Wilcoxon Rank sum test corrected for false discovery rate was used to assess the significance of differential expression for each miR between aggressive and indolent NHL. The 14 most significantly differentially expressed miRs (p

Description: E2A-PBX1 (EP1) is a chimeric oncogenic transcription factor expressed consequent to the 1;19 chromosomal translocation in cases of acute lymphoblastic leukemia (ALL). EP1 can induce transcription of reporter genes and EP1-driven oncogenesis requires direct binding of EP1 with the transcriptional co-activator and histone acetyltransferase p300. Therefore, we hypothesized that EP1 recruits p300 and other co-activators to cis-acting regulatory elements throughout the genome thereby inducing or maintaining transcription of target genes some of which contribute to the neoplastic phenotype. Here we have used chromatin immunoprecipitation followed by next generation DNA sequencing (ChIP-seq) to identify and characterize EP1-bound sites across the genome of the t(1;19)-associated, ALL-derived cell line RCH-ACV. ChIP was performed with an anti-FLAG antibody using sheared chromatin prepared from RCH-ACV cells that stably expressed FLAG-tagged EP1; ChIP from parent RCH-ACV cells not expressing FLAG-EP1 served as a negative control for peak calling. Parallel immunoprecipitations were performed with antibodies for p300 and the chromatin marks H3K4me3, H3K4me1 and H3K27me3. Sequencing of DNA purified from the immunoprecipitated material and of total RNA (RNA-seq) was carried out commercially by BGI whereas bioinformatic analyses were performed in-house. Bioinformatic analysis of data from replicate samples identified 3166 EP1 binding peaks across the RCH-ACV genome (irreproducible discovery rate threshold

Description: Abstract 1393 The oncoprotein E2A-PBX1 is produced by t(1;19)(q23;p13) in pre-B acute lymphoblastic leukemia (ALL). Perplexingly, in vitro and in vivo models of E2A-PBX1 leukemogenesis to date generate either myeloid or T-lymphoblasts, neither of which are representative of the human disease. Furthermore, we have observed recently that lineage-depleted (lin-) murine bone marrow cells infected with an E2A-PBX1 retrovirus selectively fail to repopulate the B-lymphoid compartment on transplantation into irradiated recipients. Therefore, we have now begun to investigate the mechanism by which enforced expression of E2A-PBX1 antagonizes B-lymphopoiesis. We show that E2A-PBX1-transduced lin- fetal liver progenitor cells (FLPs) fail to differentiate to committed CD45R+ pro-B-cells when cultured with IL-7 in the presence of OP9 stromal cells. Instead these cells manifest a relatively immature immunophenotype (CD45R-, CD11b-, CD117+), are murine stem cell factor (SCF) dependent, and undergo myeloid differentiation upon stimulation with granulocyte macrophage colony-stimulating factor (GM-CSF) or transplantation into irradiated mice. Amino acid substitutions in E2A-PBX1 that impair co-activator recruitment or DNA binding completely rescue the differentiation block, implying a critical role for target-gene regulation by E2A-PBX1. E2A-PBX1-transduced cells fail to induce the B-lymphoid commitment genes Ebf1 and Pax5, and chromatin immunoprecipitation (ChIP) analysis shows aberrant persistence of the Polycomb silencing mark H3K27me3 at these promoters. Previous studies have identified the Polycomb gene Bmi1 as a candidate E2A-PBX1 target gene. However, enforced expression of Bmi1 neither blocked induction of Ebf1 or Pax5 nor impaired B-lymphopoiesis, indicating that Bmi1 is not sufficient to mediate blocked differentiation by E2A-PBX1. Gene expression microarray analysis identified cytokines (ex., Csf2, IL1a, Il6) and transcription factors (HoxA9, Tal1) up-regulated in E2A-PBX1-transduced FLPs that are known to antagonize B-lymphoid development. In summary, our results support the notion that enforced expression of E2A-PBX1 in early hematopoietic progenitors blocks B-lymphopoiesis, to an important degree, by inducing the transcription of genes whose products antagonize early steps of B-lymphoid commitment, including induction of Ebf1 and Pax5. HoxA9 is particularly interesting as a mediator of E2A-PBX1 effects in view of its extreme induction ratio in our system (at least 1000-fold relative to controls), its propensity to promote myeloid over lymphoid differentiation, and its well-established role in leukemogenesis. Studies to investigate a potential requirement for HoxA9 and other candidates in mediating the E2A-PBX1-driven B-lymphoid differentiation block are ongoing. Disclosures: No relevant conflicts of interest to declare.