ALBERT

Description: Screening for copy number alterations (CNA) has improved by applying genome wide microarrays, where SNP-arrays also allow analysis of loss of heterozygosity (LOH). Currently, comparisons of high resolution microarray platforms are few, thus we performed a study to evaluate the power of differently designed microarrays for copy number analysis and LOH. We here analyzed 10 diagnostic chronic lymphocytic leukemia (CLL) samples (five IGVH mutated and five IGVH unmutated) using four different high-resolution platforms: BAC-arrays (32K), oligonucleotide-arrays (185K, Agilent), and two SNP-arrays (250K, Affymetrix and 317K, Illumina). Comparison of copy number data showed that the platforms are concordant in terms of detecting large CNA, including the known recurrent alterations. Mono-allelic and bi-allelic loss of 13q14 (3 and 1 sample, respectively), mono-allelic loss of 11q (1 sample), trisomy 12 (2 samples) and mono-allelic loss of 17p (2 samples) were concordant in all platforms. These aberrations were validated with FISH, which in addition identified subclones with mono-allelic loss of 13q14 in two cases, only detected with the BAC platform, rendering a cut-off for the power of detecting subclones to approximately 25% of investigated cells. As expected, all poor prognostic aberrations were detected in patients carrying unmutated IGHV genes whereas four of five mutated samples were detected with mono-allelic loss of 13q14, as the only recurrent alteration. Furthermore, detection of small CNA were in many cases discordant between platforms. Therefore, we defined alterations identified by at least two platforms and identified 47 losses and 31 gains using this criterion. We are currently validating the presence of a number of these alterations using other techniques. Evaluation of LOH showed concordance for 86 regions between the Illumina and Affymetrix platforms. Of these regions 12 LOH coincided with CNA, leaving the remaining 74 as copy-neutral LOH. In conclusion, all platforms investigated are powerful tools for screening of CNA, however, since non-overlapping CNA were detected by individual platforms, we emphasize the importance of validating findings. Also, there is a cut-off for detecting subclones, here estimated to 25%. Genomic arrays will improve the detection of new recurrent aberrations, which may potentially refine the prognostic hierarchy established by FISH.

Description: Abstract 296 Background: In diffuse large B-cell lymphoma (DLBCL), a number of studies have shown that dual disruption of the p16/Rb and the ARF/p53 pathway, e.g. deletion of the INK4A/ARF locus, are negative prognostic factors for survival. Molecular studies have shown that the p53-miR34 axis may be another link between the ARF/p53 pathway and the p16/Rb pathway. In a complex circuit, p53 promotes transcription of MIR34A and MIR34B/C, and the miR34s' in turn act as mediators of p53 signaling. In addition, miR34s' have been shown to inhibit MYC and several proto-oncogens that counteract the p16/Rb tumor suppressor pathway. These observations place the miR34s' at the center of cell cycle and apoptosis regulation, and loss of miR34 expression has been associated with adverse response to therapy in several tumor types. In CLL it has been suggested that miR34A expression is a surrogate marker for TP53 disruption and the MDM2 SNP309 GG genotype, both associated with a poor prognosis. Design and methods: In CD19+ B-cells isolated from healthy donors (PBL-B) and reactive lymph nodes, the expression of the –5p and –3p species of miR34A, miR34B, and miR34C was analysed by qRT-PCR using LNA-based primers from Exiqon that discriminate the individual miR34 members. Histone H3K4 and H3K27 methylation status at the MIR34A and MIR34B/C promoters was analyzed by ChIP. In 120 newly diagnosed DLBCLs we studied the combined status of TP53 mutations, MIR34A and MIR34B/C promoter DNA methylation, and the MDM2SNP309 GG genotype using a panel of PCR based methods. Results: We initially determined the expression of the individual miR34 family members in PBL-B and in reactive lymph nodes. While miR34A-5p was expressed at low levels in normal PBL-B, no expression of miR34A-3p, miR34B-5p, miR34B-3p, miR34C-5p, and miR34C-3p was observed. In reactive lymph nodes the expression of miR34A-5p was induced in average 70 fold (P

Description: Abstract 1600 Introduction: Although rare, ocular adnexal lymphomas (i.e. lymphoma of the orbit, eyelids, conjunctiva, lacrimal gland and lacrimal sac), are among the most common malignant tumors involving the ocular adnexal regions. The main subtypes are low-grade mucosa associated lymphoid tissue (MALT) lymphoma and aggressive diffuse large B cell lymphoma (DLBCL). In rare cases low-grade MALT lymphoma are reported to transform to DLBCL. It is unclear, however, which genetic events distinguish low-grade disease from aggressive, potentially fatal, disease. Material and methods: A total of 18 MALT lymphomas and 25 DLBCLs involving ocular adnexal sites were included in the study. All sections were analyzed immunohistochemically by two independent pathologists (ER, SH) using the following panel of antibodies: bcl-2, bcl-6, CD3, CD5, CD10, CD20, CD23, CD79α, cyclin D1, MUM-1 and Ki-67. Confirmed cases of DLBCL were categorized as either germinal centre B-cell-like (GCB) or non-GCB types according to the algorithm by Hans et al. Using LNA-based arrays from Exiqon, we performed global miRNA expression profiling of RNA extracted from formalin-fixed paraffin-embedded (FFPE) tissue. The most differentially expressed microRNAs were confirmed by RT-qPCR analyses. Microarray processing was performed using the R environment. Results: Of the18 MALT patients (pts.) 15 pts. (83%) presented with Stage I lymphoma and 3 pts. (17%) had Stage IV. The 5-year overall survival for the entire population was 77%. In the DLBCL group 13 pts. (52%) presented with Stage I lymphoma, 3 pts. (12%) Stage II lymphoma, 1 pt. (4%) Stage III and 8 pts. (32%) presented with Stage IV lymphoma. Nine of the DLBCLs were classified as GCB and 16 as non-GCB type. The 5-year overall survival for the entire group was only 13%. Our miR arrays and confirmatory qPCR analysis revealed 43 miRNAs with significantly altered expression profiles (41 down- and 2 upregulated) in DLBCL compared to MALT lymphoma. Seven of the miRNAs down-regulated in DLBCL relative to MALT lymphoma showed enrichment for a direct transcriptional repression by the oncoprotein MYC. Supervised hieracical clustering analysis identified tree clusters: Cluster 1: MALT (high expression), cluster 2: DLBCL (intermediate expression), cluster 3 DLBCL (low expression). Thus, apparently the DLBCLs in cluster 2 seem to resemble MALT more than DLBCLs in cluster 3. We also report loss of miRNAs involved in the regulation of NFKB1 and DNA methyltransferases in DLBCL vs MALT. Conclusion: We conclude that fundamental differences in miRNA expression exist between ocular adnexal MALT lymphoma and DLBCL. Among the possible consequences of altered miRNA expression are increased NF-kB signaling and DNA hypermethylation. However, in line with the recent observations in gastric MALT/DLBCL transformation1, we suggest the differences may at least in part be caused by MYC transcriptional regulation of miRNAs in aggressive cases. The fraction of DLBCL that is reported to arise from MALT is exceedingly low. However, in the current study we find a group of DLBCLs (cluster 2), whose level of MYC regulated miRNA expression is intermediate between MALT (cluster 1) and DLBCL (cluster 3). Thus it could be speculated whether cluster 2 DLBCLs have developed secondary to a preceding MALT lesion, which, however was not detected by histology. Disclosures: No relevant conflicts of interest to declare.

Description: Introduction Cutaneous T-cell lymphomas (CTCL) are rare malignancies, which present in the skin and presumably arise from malignant transformation of T-cells normally destined to home to the cutaneous environment. MicroRNAs (miRs) regulate gene expression at the post transcriptional level. Many studies have shown that altered miR expression is a central event in lymphomagenesis, and that miRs have potential as both diagnostic and predictive tumor markers. In CTCL we have previously identified and validated a 3 miR classifier that distinguishes CTCL from BID with 〉 95% accuracy, based upon the up-regulation of miR-155 combined with the down-regulation of miR-203 and miR-205. In normal adult tissues, miR-203 is mainly associated with keratinocyte differentiation, acting to repress stemness, and to induce cell cycle arrest and differentiation. In cancer, miR-203 has been shown to hold tumor suppressor properties, and may be down-regulated by promoter hyper-methylation. The function and implications of miR-203 for CTCL has not previously been described. In this study we have investigated the regulation and function of miR-203 in primary CTCL biopsies and cell lines. Materials and Methods Twenty-one fresh frozen primary CTCL biopsies, IL-2 independent CTCL cell lines (MyLa2059 and PDB2B), and the IL-2 dependent CTCL cell lines (SeAx and SeZ4) were analyzed in this study. Promoter methylation was analyzed by methylation specific melting curve analysis. Cell lines were transfected by electroporation of miR-203 mimic or non-template-control (mirVana, Ambion). Proliferation was measured by 3H-Thymidine and apoptosis by MMT assays. MiR-203 mimic and mock transfected cells were examined by Affymetrix RNA expression arrays (GeneChip Human Gene 2.0 ST). IL2Rβ mRNA expression was confirmed by qPCR and IL2Rβ protein levels by flow cytometry as measured by CD122 (IL2Rβ-chain), compared to CD25 (IL2Rα-chain) and CD132 (IL2Rγ-chain). Cloning was done according to the manufacturers’ recommendation (In-Fusion, Clontech) and luciferase reporter assays were performed using the Dual-Glo system (Promega). Results We show that miR-203 is epigenetically silenced by DNA methylation in both CTCL cell lines and in 9 of 21 (43%) of primary CTCL samples, and that miR-203 can be up-regulated by the hypo-methylating agents 5-azacytidine and 5-aza-2-deoxycytidine in vitro. We also show, that forced miR-203 expression in CTCL cells targets known oncogenes such as p63, Survivin and CREB. Furthermore, it is shown that induction of miR-203 reduces cell viability and decreases proliferation. mRNA array analysis of miR-203 mimic and mock transfected cells lead to the identification of 19 significantly de-regulated genes (P2), including the as yet unrecognized miR-203 target molecule IL2Rb, which is essential for IL-2 induced JAK/STAT signaling. qPCR and FACS analysis confirmed this up-regulation both at the mRNA and protein level. The IL-2 dependent cell line SeAx showed significantly more profound down-regulation of IL2Rβ upon in miR203 transfected cell lines. Preliminary luciferase reporter assays confirm that IL2Rβ expression is regulated by miR-203, providing novel evidence that miR-203 may act in concert with IL-2/STAT in CTCL pathogenesis. These experiments are currently being validated. Conclusion We provide the first evidence that miR-203 acts as a tumor suppressor in CTCL. Furthermore we show that down-regulation of miR-203 leads to increased expression of an as yet unidentified target gene, IL2Rβ, which is directly involved in JAK/STAT signaling, that plays an essential role in the regulation of T-cell proliferation. Thus, we suggest that epigenetic miR-203 down-regulation and IL2Rβ up-regulation are important early and driving events in CTCL pathogenesis. Disclosures No relevant conflicts of interest to declare.

Description: Background Mantle cell lymphoma (MCL) is a subtype of non-Hodgkin`s lymphoma (NHL) with a variable but often aggressive clinical course. The majority of MCL patients ≤65 years will experience clinical relapse during a 10-year period (Geisler et al. Br J Haematol 2012). Risk-stratification is therefore of great importance in order to identify patients who are eligible for novel or alternative treatment regimens. Minimal residual disease (MRD) monitoring can predict clinical progression in MCL and guide pre-emptive treatment with rituximab as single agent. It has thus far not been possible to predict post-treatment molecular relapse (MRD-positivity). Although some recent studies have shown that aberrant miRNA expression delineate pathogenic molecular pathways and predict survival in MCL patients, miRNA profiling has not been performed in the context of MRD and molecular relapse. Aims We assessed MCL miRNA expression, in a large, prospective, uniformly treated patient cohort followed with molecular markers for MRD to determine if miRNAs could predict molecular relapse. Methods Diagnostic MCL tumor samples from 114 patients in the Nordic MCL2 and MCL3 clinical trials were retrieved. All patients had confirmed CyclinD1 overexpression and 96 patients had Ki-67 expression measurement for use in the MIPI-B predictive score. All patients received almost identical induction treatment with six alternating cycles of maxi-CHOP and high-dose Ara-C in combination with rituximab. Patients in remission were consolidated with high dose chemotherapy followed by autologous stem cell transplantation. All patients had a molecular marker (PCR detectable t(11;14) or clonal IgH rearrangement) for MRD. The median follow-up was 6.4 years for the MCL2 cohort and 3.7 years for the MCL3 cohort. 19 miRNAs, previously found to have prognostic significance in MCL (Husby et al EHA 2014), were measured by qRT-PCR and analyzed in concordance with MRD-data. The main endpoint was first event of molecular relapse (MRD-positive sample). Results Of the 114 patients in the study, 71 (62%) patients became MRD-positive in the follow-up period. Of the 19 examined miRNAs, 10 miRNAs showed unique qPCR melting curves and were analyzed with respect to MRD. Four miRNAs (miR-92a, miR-3687, miR-486-5p and miR-185-5p) were significantly up-regulated in patients who had molecular relapse (t-test; respectively p = 0.010, p = 0.019, p = 0.048, p = 0.043). However miR-18b, previously identified as prognostic marker regarding survival, was not significantly overexpressed. We hereafter investigated if a newly derived prognostic score, the MIPI-B-miR, which incorporates miR-18b with the MIPI-B, could predict molecular relapse. The MIPI-B-miR high-risk patients had significantly shorter time to first molecular relapse than MIPI-B high-risk patients (Figure 1). Functional studies of these aberrantly expressed miRs are ongoing. Conclusion Patients with molecular relapse had significantly increased levels of miR-92a, miR-3687, miR-486-5p and miR-185-5p, and MIPI-B-miR improved MRD prediction compared to MIPI-B. Aberrant miRNA profiles may be able to predict molecular relapse, and may already at diagnosis identify patients eligible to anti-CD20 antibody maintenance or alternative regimens. However validation in other cohorts is needed. Figure 1 Figure 1. Disclosures Kolstad: Nordic Nanovector: Membership on an entity's Board of Directors or advisory committees, Research Funding. Räty:GlaxoSmithKline Ltd: Honoraria; Roche Ltd: Membership on an entity's Board of Directors or advisory committees; Mundipharma: Honoraria.

Description: Abstract 1364 Introduction: Cytosine methylation (mC) is a major DNA modification in higher eukaryotic genomes, which is involved in transcriptional silencing. A large amount of data has shown that patterns of DNA methylation are perturbed in hematological cancers including diffuse large B-cell lymphoma (DLBCL). The discovery that the TET hydroxylases convert mC to hydroxymethylcytosine (hmC) is a major break through for our understanding of how DNA methylation is deregulated. Multiple reports describe TET2 (Ten-Eleven Translocation 2) loss-of-function mutations in myeloid malignancies, and a recent study shows that TET2 inactivation perturbs both myeloid and lymphoid development in the mouse, and identifies TET2 mutations in ∼2% of human B-cell lymphoma (Quivoron et al, Cancer Cell 20, 1–14, 2011). Aims: In the present study our aims are to determine the frequency and clinical impact of TET2 mutations in DLBCL, to identify TET2 target genes in CD34+ cells, normal- and malignant B-cells, and evaluate the role of TET2 mutations on the methylation pattern at TET2 targets genes in normal and malignant hematopoiesis. Methods: DNA was isolated from fresh frozen DLBCL (n=110), normal CD34+ cells and B-cells, and a TET2 mutant DLBCL-cell line. Mutation scanning was performed by denaturing gradient gel electrophoresis (DGGE) and automated sequencing. Global methylation profiling was done by Illumina Infinium microarrays, methylation at individual genes by methylation specific melting curve analysis and pyrosequencing. Global mC and hmC patterns were determined by DNA immunoprecipitation and promoter array analysis in cell lines, B-cells and CD34+ cells. TET2 target genes were identified by ChIP followed by deep sequencing. Gene expression by Nimblegen custom made arrays and RT-qPCR. Results: We identified TET2 mutations in 15% of primary diffuse DLBCL, including missense mutation in the catalytic domain (n=8, 2 of which showed allelic loss), loss-of-function mutations (n=7, one of which showed allelic loss), and missense mutation outside the catalytic domain (n=1 with allelic loss). Somatic origin of these mutations was verified in 11 of the 16 cases where matched normal tissue was available. No difference in overall survival was observed between TET2mut and TET2wt cases (P=0.17). To a large extent, the TET2 targets genes identified by ChIP seq analysis were overlapping in CD34+ cells, normal- and malignant B-cells. Gene ontology analysis showed that TET2 target genes are mainly involved in DNA metabolism and repair, metabolic processes and cell cycle homeostasis. Global methylation in TET2mut and TET2wt cases and gene expression data are being analyzed in DLBCL samples. In addition, the distribution patterns of hmC and mC at TET2 target genes and the relation to gene expression is being analyzed in a TET2 mutant DLBCL cell line, normal B-cells and CD34+ cells. Conclusion and further analyses: Here, we show that TET2 mutations are frequent in DLBCL, and identify the TET2 target genes in CD34+ cells, and in normal and malignant B-cells. The role of TET2 mutations for global methylation and for the methylation patterns at TET2 target genes will be presented at the meeting. By investigating the clinical implications of TET2 mutations we aim to identify DLBCL subsets that may benefit from hypomethylating therapy. Furthermore, the identification of hypermethylated TET2 target genes will hopefully contribute to molecular understanding of how TET2 mutations induces malignant transformation. Disclosures: Christensen: EpiTherapeutics: cofounder of EpiTherapeutics and have shares and warrants in the company. Helin:EpiTherapeutics: cofounder of EpiTherapeutics and have shares and warrants in the company.

Description: Cutaneous T-cell lymphomas (CTCLs) are the most frequent primary skin lymphomas. Nevertheless, diagnosis of early disease has proven difficult because of a clinical and histologic resemblance to benign inflammatory skin diseases. To address whether microRNA (miRNA) profiling can discriminate CTCL from benign inflammation, we studied miRNA expression levels in 198 patients with CTCL, peripheral T-cell lymphoma (PTL), and benign skin diseases (psoriasis and dermatitis). Using microarrays, we show that the most induced (miR-326, miR-663b, and miR-711) and repressed (miR-203 and miR-205) miRNAs distinguish CTCL from benign skin diseases with 〉 90% accuracy in a training set of 90 samples and a test set of 58 blinded samples. These miRNAs also distinguish malignant and benign lesions in an independent set of 50 patients with PTL and skin inflammation and in experimental human xenograft mouse models of psoriasis and CTCL. Quantitative (q)RT-PCR analysis of 103 patients with CTCL and benign skin disorders validates differential expression of 4 of the 5 miRNAs and confirms previous reports on miR-155 in CTCL. A qRT-PCR–based classifier consisting of miR-155, miR-203, and miR-205 distinguishes CTCL from benign disorders with high specificity and sensitivity, and with a classification accuracy of 95%, indicating that miRNAs have a high diagnostic potential in CTCL.

Description: Key Points miR-18b overexpression identified patients with poor prognosis in 2 large prospective homogenously treated MCL cohorts. miR-18b overexpression adds prognostic information to the MIPI-B prognosticator.