ALBERT

Description: Background Follicular lymphoma (FL) is a B-cell lymphoma whose cytogenetic hallmark is the translocation t(14;18)(q32;q21) which juxtaposes the BCL2 oncogene to the immunoglobulin heavy chain locus (IGHV). FLs maintain key features of normal germinal center reactions, such as ongoing somatic hypermutation (SHM) of IGHV genes and selection for a functional B-cell receptor. SHM is mediated by activation-induced cytidine deaminase (AID) leading to single nucleotide exchange in IGHV genes and to a much lesser extent in non-IG genes. It was our objective to investigate the clonal evolution of t(14,18) FL from primary to relapse tumors simultaneously on several genetic and epigenetic levels. Methods We studied paired primary and relapsed tumors from 33 patients with t(14;18)-positive FL (76 samples: 25 pairs; 6 trios and 2 quadruples). In a core set of 19 patients we performed Sanger and next generation sequencing of the clonal VHDHJH rearrangements of the IGHV locus. We performed deep sequencing of 9 genes (BCL2, BCL6, MYC, RHOH, PAX5, IRF4, C2TA, REL and PIM1) targeted by aberrant SHM (aSHM) in lymphoma for 69 FL samples including the complete core set. We furthermore analyzed mutations in the coding regions of 10 candidate driver genes of lymphomagenesis (BCL2, MLL2, CREBBP, TNFRSF14, EZH2, EP300, MEF2B, BCL6, MYC, TP53) by deep sequencing. In addition to genetic analyses, evolutionary patterns of DNA-methylation were addressed by Illumina 27k arrays. Results We found strong evidence for ongoing selection against replacement mutations in the IGHV genes both in complementarity determining regions and framework regions, consistent with ongoing dependence of FL on a functional B-cell receptor and stimulation by antigens. Using mean normalized Hamming distance as a quantitative measure for the evolutionary divergence of paired samples (IGHV-divergence) and analyzing phylogenetic trees we classified the patterns of evolution into 3 categories: “No Evolution” (shared IGHV sequences in primary and relapse tumors), “Sequential Evolution” (relapse sequences emerge out of primary ones), “Divergent Evolution” (sequences of primary and relapse sample appear disjoint). We observed a mutation frequency of 62.0 per 100 kb in aSHM targets. These mutations were strongly enriched at the WRCY/RGYW target motifs characteristic for the SHM/AID machinery (OR=3.4; p

Description: The class of the so-called long non-coding RNAs (lncRNAs) is defined by their lacking coding potential and their length of more than 200 nucleotides. LncRNAs are readily detected in transcriptome analysis but have so far not been characterized or catalogued exhaustively. Although lncRNAs make up a major portion of the mammalian non-coding transcriptome, it is challenging to predict their functions due to their diversity in terms of size, sequence and biogenesis. However, several lncRNAs have already been shown to be involved in crucial cellular processes such as imprinting, regulation of cell proliferation and cellular differentiation. Their mode of action generally involves epigenetic modifications as well as transcriptional and post-transcriptional regulation. Within the ICGC (International Cancer Genome Consortium)-MMML-Seq (Molecular Mechanisms in Malignant Lymphoma by Sequencing) Consortium, which aims at fully characterizing a total of 250 germinal center derived B-cell lymphomas, including Burkitt lymphoma (BL), diffuse large B-cell lymphoma (DLBCL) and follicular lymphoma (FL), we conducted a search for yet unidentified genes. We here report the identification of a novel transcript, which we have named BLUT (Burkitt Lymphoma Unknown Transcript). BLUT is highly expressed in BL, with only very low expression levels in FL and no detectable expression in DLBCL. It is around 1050 nucleotides long with splice evidence for three exons, whereby exons 1 and 3 are shared between all isoforms. BLUT represents a putative lncRNA since it lacks conserved protein domains and has a splice site conservation pattern typical of non-coding transcripts. Furthermore, it is predicted as non-coding by the software Coding Potential Calculator and there was not a single peptide match between a published peptide database (Human Proteome Map) and any potential BLUT peptide (all isoforms). Moreover, BLUT shares a high sequence identity with two (so far) uncharacterized non-coding RNAs (Pan Troglodyte, Chlorocebus sabeus). We tested a cell line panel (consisting of 16 different cell lines including non-lymphoma cell lines) for BLUT expression but could only detect it in the two BL cell lines Namalwa and Raji. Cellular fractionation experiments showed that BLUT is predominantly localized in the nucleus in both Namalwa and Raji. Overexpression of BLUT in SU-DHL-4 resulted in the identification of 222 differentially regulated genes (FDR 〈 0.05) with a logFC of ± 0.25, among which 49 genes had a logFC of ± 0.50. Several lymphoma relevant genes were part of this list including BIN1, CCL22, CCR7, EBI3, ID2, JAG1, NQO1, and RGS1. Given its nuclear localization, we currently perform chromatin isolation by RNA purification experiments to identify the genomic regions which BLUT binds to. In combination with our overexpression data this will allow further insights into the functions of BLUT. In summary, we have identified a novel long-noncoding RNA termed BLUT, which is differentially expressed between lymphoma subtypes with a high expression in Burkitt lymphoma. Overexpression of BLUT resulted in the deregulation of several genes with known roles in lymphomagenesis. Given its predominantly nuclear localization, we are currently investigating its functions on chromatin regulation. (Supported by BMBF through 01KU1002A-J and by the Duesseldorf School of Oncology (funded by the Comprehensive Cancer Center Duesseldorf/Deutsche Krebshilfe and the Medical Faculty HHU Düsseldorf)) Disclosures Truemper: Amgen, roche, Mundipharma: Research Funding; Sandoz, Celgene, AMGEN, Nordic Nanovector: Other: Advisory board.

Description: The discovery of microRNAs (miRNAs), a group of small non-coding regulatory RNAs, revolutionized the field of posttranscriptional gene regulation. Mature miRNAs are single-stranded RNA molecules of 20- to 23-nucleotide length that control gene expression. They typically reduce the translation and stability of mRNAs important in tumorigenesis mediating processes such as inflammation, cell cycle regulation, stress response, differentiation, apoptosis, and invasion. Germinal center (GC) derived B-cell lymphomas, including Burkitt lymphoma (BL), diffuse large B-cell lymphoma (DLBCL) and follicular lymphoma (FL), are the most frequent malignant lymphomas. Although clear distinctions on histologic and genetic grounds exist, there is also a large number of cases with intermediate features, not unequivocally attributable to one of these entities. Recently, a signature of 38 miRNAs containing MYC regulated and nuclear factor-kB pathway-associated miRNAs was published, which differentiated BL from DLBCL. However, available data is preliminary as published profiles are not derived from large sample collections and also originate mostly from PCR-based approaches and microarrays. Yet, only sequencing-based approaches allow for an unbiased analysis and the discovery of novel miRNAs and small RNA classes. The ICGC (International Cancer Genome Consortium)-MMML-Seq (Molecular Mechanisms in Malignant Lymphoma by Sequencing) Consortium aims at fully characterizing a total of 250 GC derived B-cell lymphomas over a period of five years. We here report the miRNA analysis of the first 49 patient samples including BL, DLBCL and FL using Illumina technology. We are recording the classical miRNA fraction (18-35 nucleotides) as well as a larger size fraction (35-90 nucleotides). Initial differential expression analyses comparing BL against non-BL showed eight miRNAs to be differentially expressed. This comprises hsa-miR-150, which has been ascribed a role in the adaptive immune response and which was shown to be upregulated in cutaneous marginal zone B-cell lymphomas. Further differentially expressed miRNAs include e.g. hsa-miR-211, hsa-miR-548ac and hsa-miR-1244-1/3, which represent novel targets in lymphomagenesis. Among the many bioinformatically predicted novel miRNAs, we have been able to validate four candidates by Northern Blot experiments and are currently performing functional studies. Mutational analysis is ongoing, but so far has yielded two miRNAs with mutations in the mature sequences (hsa-miR-4537, hsa-miR-758). In conclusion, our initial series of 49 cases from the ICGC-MMML-Seq cohort has already given exciting insights into the role of miRNAs in GC derived B-cell lymphomas. (Supported by BMBF through 01KU1002A-J) Disclosures: No relevant conflicts of interest to declare.

Description: Background and Aims A novel genetic subtype of B cell precursor acute lymphoblastic leukemia (B-ALL) is characterized by rearrangement of NUTM1 (NUTM1r) on 15q14 resulting in fusion of NUTM1 to one of several partner genes such as CUX1, ACIN1, BRD9, and IKZF1. The downstream effects of NUTM1r include upregulation of the proto-oncogene BMI1 and specific fusions also induce transcription of the HOXA gene cluster (Hormann et al. Haematologica 2019; Li et al. PNAS 2018). This novel subtype is rare in children, but appears to be more prevalent among infants negative for KMT2A rearrangement (KMT2Ar) based on the frequency of karyotypic 15q aberrations (De Lorenzo et al. Leukemia 2014). This international collaborative study aimed to determine the frequency of NUTM1r in infant and pediatric cohorts, and to characterize the demographic, clinical and molecular features of NUTM1r-positive B-ALL. Patients and Methods Interfant-related study groups provided NUTM1 screening results for KMT2Ar-negative Interfant-99 and -06 cases with karyotypic 15q aberration, normal karyotype, or missing karyotype. Additionally, NUTM1r-positive cases of any age were collected from the study groups united in the Ponte di Legno consortium. The identified NUTM1r-positive children were diagnosed between 1995-2019, infants (≤365 days of age) included in the Interfant-99 or 06 trials were diagnosed between 2000-2016, and remaining infants between 1986-2019. The techniques used for the detection of NUTM1r were break-apart FISH, RNA sequencing, and RT-PCR. Event-free survival (EFS) and overall survival (OS) were estimated according to Kaplan-Meier, standard error according to Greenwood, and the curves were compared by log-rank test. Results We identified 81 NUTM1r cases, including 35 Interfant-enrolled infants, 10 other infants and 36 children. NUTM1r was reported to be rare among pediatric B-ALL with an estimated frequency range of 0.28-0.86%. The median age among NUTM1r-positive children was 4.5 years (range 1-15). Among KMT2Ar-negative infants the frequency of NUTM1r was 21.7%. Of NUTM1r-positive infants, 54% were