Stockholm, Sweden, June 13 – 16, 2013

Aims: To gain insight of the unusual localisation of these lymphoma, we Non-Hodgkin Lymphoma - Biology analysed the immunoglobulin (IG) repertoire of 55 cases of PIOL, the largest series to date. Methods: Monoclonal IG heavy chain rearrangements, and for a fraction of cas - P281 es also chain rearrangements, were sequenced from PCR products obtained by amplification using either framework 1 or peptide leader primers. BMI1, THE POLYCOMB-GROUP , IS RECURRENTLY REARRANGED In a few cases sequencing was performed after subcloning of PCR products IN PROGRESSIVE/TRANSFORMED CLL AND MCL generated with high fidelity Taq polymerase. Comparison with germline Wlodarska 1,* L Rouhi 1, J Ferreiro 1, N Put 1, T Tousseyn 2, C Lefebvre 3, A Gar - 4 1 5 6 7 1,8 sequences was done using IMGT data and tools. diner , W De Kelver , H Demuynck , J Verschuere , I Theate , C Vicente , Results: We observed a highly restricted IGHV usage, with a single gene, L Michaux 1, J Cools 1,8, P Vandenberghe 1 1 2 namely IGHV4-34, present in 69.1% (38/55) of cases. The second most fre - Center for Human Genetics, Department of Pathology, KU Leuven, Leuven, quent gene was IGHV3-7, utilized in 7.3% (4/55) of cases. Thus, only two IGHV Belgium, 3Department of Oncogenetics, CHU Albert Michallon, Grenoble, 4 accounted for more than three quarters of the PIOL IGHV repertoire. Bias France, Department of Hematology, Royal Bournemouth Hospital , in IGHD and IGHJ genes were also observed, although to a lesser extent, with Bournemouch, United Kingdom, 5Department of Hematology, Hospital of Roe - 6 an underrepresentation of IGHD6 subgroup genes and a high IGHJ5 / IGHJ6 selare, Roeselare, Department of Hematology, Hospital of Ronse, Ronse, ratio as compared to other B-cell malignancies. Heavy complementarity-deter - 7Department of Pathology, Cliniques Universitaires Saint-Luc, Brussels, 8 mining region 3 (VH CDR3) were short (median length 14 amino acids) and in Department of Molecular and Developmental Genetics, VIB, Leuven, Belgium half of cases (28/55) carried electropositive residues with predicted isoelectric values of 7.0 or greater (up to 13.0). Remarkably, 3 of the 4 cases expressing Background: Chronic lymphocytic leukemia (CLL) has a variable clinical the IGHV3-7 gene had 11 aminoacid-long, electropositive VH CDR3s with course, ranging from a very indolent to a rapidly progressing disease. In up to shared motifs, which could be considered as “stereotyped” antigen-binding 10% of CLL patients, transformation to highly aggressive and usually fatal lym - sites. Except for two unmutated cases, all sequences had a high number of phoma (Richter syndrome) has been reported. Although several factors predis - somatic hypermutations (SHM) as the mean % of identity from their germline posing CLL to a high grade transformation and/or chemoresistance have been counterpart was 86.7%. Analysis of the distribution of SHM in the subgroup of recently identified (e.g. mutations of NOTCH1, SF3B1 and BIRC3 ), molecular PIOL cases utilizing the IGHV4-34 gene revealed high replacement (R) to silent events associated with Richter transformation remain largely unknown. How - (S) mutation ratios in CDRs (R/S>3.0) along with low R/S ratios in FRs. Iden - ever, accumulating evidence indicate that this process may be conducted by tical replacement mutations (“stereotyped” amino acid changes) at certain several oncogenes, including MYC, BCL3 and NOTCH1 , activated by IG -relat - codon positions were identified amongst rearrangements utilizing the IGHV4- ed translocations acquired during clinical course of disease. 34 gene, some of which were distinct from those previously reported for IGHV4- Aims: Our study aimed at molecular characterization of the novel 34 rearrangements in other B cell malignancies. In addition, the IGVH4-34 spe - t(10;14)(p12;q32)/t(10;22)(p12;q11) identified in six cases of progressive/ trans - cific motif responsible for binding in superantigenic fashion the N-acetyllac - formed CLL, and various 10p11p13 rearrangements recurrently observed in tosamine antigenic determinant was altered in a minority of sequences mantle cell lymphoma (MCL). (6/38,15.8%); however, the most critical residue of this motif (TRP at position Methods: The work was performed using FISH, qRT-PCR, IHC, high-resolu - 7 in FR1) was intact in all 38 cases. Sequencing of multiple (at least 20) clones tion array CGH (aCGH), Sanger sequencing and RNA-sequencing. in 9 cases of PIOL (including 6 IGHV4-34 cases) showed intraclonal diversity Results: (i) Six cases of progressive/transformed CLL with the IGH - or IGK - in all of them. Finally the repertoire of IG light chains was also biased for IGHV4- involving translocations targeting 10p12 were collected. Molecular profiles of 34 PIOL as 7/23 cases (30.4%) had a IGKV3-20 / IGKJ1 rearrangement. Iden - these leukemias were heterogeneous (mutated or unmutated VH, presence of tical amino-acid replacements resulting from SHM were also observed among either del(11q) or del(13q14), or trisomy 12) and the leukemia cells were neg - these sequences. ative for common mutations of NOTCH1 and TP53 . All patients died within 1- Summary / Conclusion: PIOL display a highly biased IG gene repertoire with 37 months after detection of t(10;14)/t(10;22). The extensive BAC-walking FISH very precise targeting and distinctive features of SHM, suggestive of selection analysis eventually mapped the 10p12 breakpoint in the region harbouring by specific (super)antigen(s) in lymphomagenesis. BMI1 gene. Upregulation of BMI1 mRNA was demonstrated by QRT-PCR analysis in one case documented by paired diagnostic/follow-up samples. (ii) 16 MCL cases with various 10p11-13 aberrations were initially subjected to P283 FISH and aCGH analysis. All the rearrangements affected BMI1 which was either duplicated/amplified (4 cases), or involved in non-reciprocal transloca - JUNCTIONAL ADHESION MOLECULE C CONTROLS PROLIFERATION, tions (9 cases), or affected by balanced translocations/insertions (3 cases). HOMING AND ENGRAFTMENT OF NORMAL AND MALIGNANT HUMAN B RNA-sequencing performed in three available cases did not identify BMI1 -relat - CELLS ed fusions/mutations, but demonstrated a significant upregulation of BMI1 , in C Doñate 1,* C Ody 2, B Imhof 2, T Matthes 1 addition to overexpressed cyclin D1 and SOX11 . 1Hematology department, University Hospital Geneva, 2Pathology and Summary / Conclusion: We show for the first time that BMI1 , the Polycomb department, University Medical Center, Geneva, Switzerland group gene and postulated lymphoma-related oncogene, is recurrently target - ed by chromosomal aberrations in two human B-cell malignancies, CLL and Background: The junctional adhesion molecules (JAMs) are a subgroup of the MCL. In the former neoplasm, BMI1 was constantly affected by IG -mediated Immunoglobulin superfamily. JAM members localize at endothelial tight junc - translocations exclusively detected at time of CLL progression or Richter trans - tions and have been involved in the formation and maintenance of inter- formation. MCL displayed a much broader spectrum of BMI1 rearrangements of endothelial junctions and in leukocyte transmigration. Earlier studies have also which the most frequent were unbalanced (non- IG ) translocations. These aber - demonstrated that tight junction molecules can regulate cell polarity and vas - rations were associated with upregulation of BMI1 by mechanisms which remain cular permeability, as well as cell proliferation and differentiation. JAM-C is also elusive. Of note, the previously identified amplification of BMI1 occurred in only expressed in human B cells and its expression is tightly controlled during dif - 40% of MCL cases with the 10p12/ BMI1 rearrangements. Collectively, our work ferentiation. Expression on malignant B cells was found to be disease-specif - identified BMI1 as a new player implicated in progression and high grade trans - ic, allowing the classification into JAM-Cpos and JAM-Cneg B-cell lymphomas. formation of CLL and confirmed its involvement in the pathogenesis of MCL. Aims: In the current study, we investigated the role of JAM-C in the prolifera - Although many solid tumors and haematological malignancies display an aber - tion, homing and engraftment of normal and malignant B cells. rant expression of BMI1, the underlying 10p12 chromosomal rearrangements Methods: Human B cells were isolated from peripheral blood of healthy donors (except of gain/amplification) have never been reported in human cancers. and lymphoma patients. To analyze the role of JAM-C in proliferation, cells were activated and cultured in the presence of anti-JAM-C antibodies, and pro - liferation was measured by flow cytometry. The signaling pathways induced by P282 binding of JAM-C antibodies were monitored at a single cell level. Using phos - pho-specific antibodies for p38, Erk 1/2, JNK (Mitogen-activated kinas - MASSIVE IMMUNOGLOBULIN REPERTOIRE BIAS IN PRIMARY INTRAOC - es, MAPK), Stat3 (Signal transducer and activator of transcription), and for Akt ULAR LYMPHOMAS SUGGESTS ANTIGENIC SELECTION OF THE NEO - (PI3K/AKT/mTOR cell survival pathway), phosphorylation profiles for each pro - PLASTIC CELLS DURING LYMPHOMAGENESIS. 1 2 3 4 5 tein were analyzed by phospho-specific flow cytometry. To investigate the role N Belhouchi , E Stalika , B Bodaghi , M Boudjoghra , N Cassoux , of JAM-C in B cell migration, B cells were incubated with six different anti-JAM- C Fardeau 5, P Le Hoang 3, H Merle-Beral 1, K Stamatopoulos 2, F Davi 1,* 1 2 C antibodies and injected i.v. into NOD/SCID mice. Homing of cells to lymphoid Hematology, Hopital Pitie-Salpetriere & UPMC Paris 6, Paris, France, Hema - organs (bone marrow, spleen, lymph nodes) was analyzed one hour later by tology and HCT unit, G. Papanicolaou Hospital, Thessaloniki, Greece, 3Oph - 4 5 flow cytometry. To investigate the role of JAM-C in lymphoma dissemination, the talmology, Hopital Pitie-Salpetriere & UPMC Paris 6, Hematology, Ophtalmol - JAM-C positive mantle cell lymphoma B-cell line Jeko-1 was used for long- ogy, Hopital Pitie-Salpetriere, Paris, France term engraftment assays. Jeko-1 cells were injected into NOD/SCID mice and animals were treated for three weeks with anti-JAM-C antibodies. Tumor bur - Background: Primary intraocular lymphoma (PIOL) is a high grade lymphoma, den was evaluated in lymphoid organs on day 26. which affects the retina,vitreous and/or the optic nerve. The vast majority of cas - Results: Incubation of normal and malignant JAM-Cpos B cells with anti-JAM- es are classified as diffuse large B cell lymphoma and considered as a subtype C antibodies significantly reduced proliferation by 30-35%. Moreover, the bind - of primary central nervous system lymphoma.

haematologica | 2013; 98(s1) | 121 18 th Congress of the European Hematology Association ing of anti-JAM-C antibodies inhibited the phosphorylation of ERK1/2 by 35%, genetic hits that occur in immortalized T-cells during the later life are essential without affecting other signaling pathways. Treatment with 2 out of 6 monoclon - for its pathogenesis. However, little has been known about those genetic hits al anti-JAM-C antibodies reduced the homing of normal and JAM-Cpos lym - that are involved in the pathogenesis of ATL. phoma B cells to lymph nodes (50%), bone marrow (30%) and spleen (65%). Aims: The purpose of this study is to understand the genetic basis of ATL, we These two antibodies recognize different epitopes on the JAM-C molecule, as performed whole exome sequencing and follow-up mutation analysis in a large demonstrated by competitive binding and Plasmon resonance assays. Long- series to confirm the findings in the exome analysis. term administration of the most efficient anti-JAM-C antibody reduced drasti - Methods: We performed whole exome sequencing of paired-tumor/normal cally the engraftment of JAM-Cpos Jeko-1 cells in the bone marrow (94%), DNA from a single case with ATL lymphoma type. In addition, mutations of spleen (100%) and lymph nodes (99%) of NOD/SCID mice. TET2 , IDH1/2, and DNMT3A were screened in an extended cohort of 145 ATL Summary / Conclusion: Despite considerable progress in the treatment of cases using targeted deep sequencing. mature B-cell lymphomas, aggressive forms still remain incurable and new Results: A total of 77 non-silent somatic mutations were detected by whole treatment strategies are needed. Our results demonstrate a functional role of exome sequencing. Among them, we identified a TET2 mutation (R1261C). JAM-C in B cell homing, proliferation and engraftment into lymphoid organs. TET2 mutations have been found in a wide variety of myeloid malignancies at We also identified for the first time the intracellular MAPK cascade as the JAM- high frequencies. The TET family of is thought to be involved in the C driven signaling pathway in JAM-Cpos B cells. Anti-JAM-C antibodies could epigenetic regulation of through catalyzing conversion of 5’- thus represent an efficient therapeutic approach reducing cell proliferation and methyl cytosine to 5’-hydroxymethyl cytosine, which are supposed to be further preventing lymphoma B cells from reaching supportive lymphoid microenvi - converted to unmethylated cytosine. One of the recent interests is frequent ronments in bone marrow, lymph nodes and spleen. mutation of TET2 , IDH2 and DNMT3A in other PTCLs, such as angioim - munoblastic T-cell lymphomas (AITL) and PTCL not otherwise specified (PTCL- NOS). So we investigated mutations of these genes in a cohort of 145 ATL. P284 In total, 17 TET2 mutations were found in 14 (9.6%) out of the 145 ATL sam - ples. Less frequent mutations of IDH2 and DNMT3A were also identified. Dif - DETECTION OF PERIPHERAL BLOOD MONOCLONAL T LYMPHOCYTO - ferent subtypes of ATL were affected with 6 out of 47 acute, 3 out of 36 chron - SIS AND RISK OF T LYMPHOPROLIFERATIVE DISEASE DEVELOPMENT ic and 5 out of 46 lymphoma types having TET2 mutations. Biallelic involve - R Di Gaetano 1,* V Gasparetto 1, C Laura 1, B Callegari 1, G Tagariello 1 1 ment was suggested in 4 out of the 14 cases. TET2 mutations seemed to have Haematology, Castelfranco Veneto Hospital, Castelfranco Veneto, Italy no significant impacts on overall survival. Less common mutations were found in IDH2 (p.R172K and p.R172T) and DNMT3A (p.G543fs and p.R882H), of Background: Occasional evidence of monoclonal gammopathy of undeter - which 2 IDH2 mutations coexisted with TET2 mutations. In deep sequencing, mined significance (MGUS) or monoclonal B lymphocytosis (MBL) are consid - it revealed that the TET2 mutations harbored a major tumor population in most ered predisposing to the development of multiple myeloma and chronic lym - evaluable cases, while the TET2 mutations in two cases only involved a minor phocytic leukemia (CLL) respectively and it is recommended to monitor them population. In some cases, TET2 mutations seemed to be among early genet - for possible early diagnosis. In cutaneous T lymphoma (CTL), such as myco - ic event in ATL, but nevertheless, it should not before HTLV-1 infection, and in sis fungoides and Sezary syndrome, has been demonstrated the presence of other cases, TET2 mutations could be relatively late events, found in a subpop - clonal T lymphocytes even in the peripheral blood. ulation. So our finding of TET2 mutations in ATL suggested that mutated TET2 Aims: It seems possible that the detection of asymptomatic clonal proliferations have unique roles in T-cells, contributing to the development of peripheral T- lymphocytosis attributable to CTL may represent a predisposing condition to cell neoplasms, most likely through epigenetic deregulation (Figure). the development of cutaneous lymphomas. Summary / Conclusion: TET2 was mutated in ~10% of ATL patients, indicat - Methods: Every year we analyze by flow cytometry about 1700 samples. ing common pathogenesis between AITL and other PTCLs. Our finding sug - Patients with peripheral lymphocytosis undergo a standard panel for T, B and gested a common role of deregulated epigenetic machinery in the development NK cells. If clonal B is excluded, we proceed to phenotypic T CD4 subsets of mature T-cell neoplasms including ATL. (CD7, CD26), analysis of V β chains and TCR when necessary. Results: From January 2010 to September 2012 we observed 28 cases of CD3 T> 80% of total lymphocytes (CD3 ≥ 3.000/mm 3). Lymphocytosis was variable between 3, 000 and 10.000/mm 3, with an average of 5, 350. 14 subjects with CD4+CD8- > 75% of T cells showed CD7 low/neg, CD26 low/neg- and TCR Vβ suggestive of monoclonality, then confirmed by molecular biology. These subjects were followed up with blood count and clinical / dermatological eval - uation twice a year in the event of increased peripheral lymphocytosis or appearance of symptoms suggestive of lymphoma. Two patients developed erythematous skin lesions and skin biopsy diagnosed the presence of Sezary syndrome, also confirmed on lymph node and bone marrow biopsies. Both these patients belonged to the subgroup with CD3 + CD4 + CD7-/ + CD8- CD26- phenotype, typical of Sezary syndrome. Summary / Conclusion: 7.1% (2/28) of patients with monoclonal T lympho - cytosis have developed a Sezary syndrome in a median follow-up of 20 months. Based on these preliminary data the occasional finding of T lymphocytosis with “atypical” phenotype CD3 + CD4 + CD7-/ + CD26-, may be a useful tool in order to allow early diagnosis of CTL in analogy with the strategy currently used for patients with MBL. If the hypothesis will be confirmed in larger cohort this approach could represent an advantage in terms of early diagnosis and time - ly treatment in this subset of patients.

Figure 1. P285 WHOLE EXOME ANALYSIS REVEALS MUTATIONS OF TET2 IN ADULT T-CELL LEUKEMIA/LYMPHOMA P286 Y Nagata 1,* A Sato-Otsubo 1, Y Okuno 1, Y Shiraishi 2, K Chiba 2, H Tanaka 2, 1 1 1 3 3 4 VITAMIN D3 AND LENALIDOMIDE SYNERGIZE TO INDUCE APOPTOSIS A Kon , K Yoshida , M Sanada , K Ishiyama , S Miyawaki , A Kitanaka , K Shi - IN MANTLE CELL LYMPHOMA VIA THE INDUCTION OF THE BH3-ONLY moda 4, S Miyano 2, T Watanabe 5, S Ogawa 1 1 2 BIK PROTEIN Cancer Genomics Project, The University of Tokyo, Laboratory of DNA Infor - C Brosseau 1,2,3,4 ,* C Dousset 1,2,3,4,5 , T Cyrille 1,2,3,4 , S Maiga 1,2,3 , mation Analysis, Center, The University of Tokyo, 3Division of 4 5 1,2,3 1,2,3,4 1,2,3,4,5 4 P Moreau , , M Amiot , C Pellat-Deceunynck , S Le Gouill Hematology, Tokyo Metropolitan Ohtsuka Hospital, Tokyo, Department of Gas - 1INSERM UMR892, 2CNRS UMR6299, 3Université de Nantes, 4Service d’Hé - troenterology and Hematology, Faculty of Medicine, University of Miyazaki, matologie, CHU, 5CIC INSERM, Nantes, France Miyazaki, 5Department of Medical Genome Science, The University of Tokyo, Tokyo, Japan Background: Targeted therapies are being tested in the MCL. Among all new treatment options, lenalidomide appears as one of the most efficient molecule. Background: Adult T-cell leukemia/lymphoma (ATL) is an aggressive form of Lenalidomide has multiple modes of action targeting the tumor cell and its envi - peripheral T-cell lymphoma (PTCL), which is etiologically associated with ronment including immune system. It is widely reported that cancer patients are human T-lymphotropic virus type I (HTLV-1) infection during early infancy. deficient in vitamin D3 (1,25-dihydroxyvitamin D3, VD3) and recent studies in Although HTLV-1 can effectively immortalize T-cells, there is a long latency lymphomas have shown that VD3 rate is significantly associated with survival. period of ~50 years prior to the onset of ATL, suggesting that HTLV-1 infection While relations between VD3 and incidence of cancer remain unresolved, it was alone may not be sufficient for the development of ATL, but additional acquired shown that VD3 contributes as an anticancer agent through its anti-prolifera -

122 | haematologica | 2013; 98(s1) Stockholm, Sweden, June 13 – 16, 2013 tive, pro-differentiation, anti-inflammatory and anti-angiogenic properties. Summary / Conclusion: Taken together, our findings clearly demonstrate an Aims: We assessed the efficacy of VD3 to potentiate cell death induced by important pathophysiological role of CXCR4 in NHL. Our model may further lenalidomide in MCL cell lines and patients’ samples and unraveled the mech - serve to elucidate CXCR4-regulated molecular events potentially involved in the anism of cell death. pathogenesis of NHL, and strongly support targeting CXCR4 as therapeutic Methods: Experiments were conducted in a panel of 6 MCL cell lines (JEKO- strategy. 1, MINO, GRANTA-519, UPN-1, REC-1 and Z138) and 8 primary samples. Results: After 6 days of treatment, MCL cells were weakly sensitive to low doses of lenalidomide (1 µM and 10µM for cell lines and samples, respective - P288 ly). Addition of physiological doses of VD3 (100nM) significantly and synergis - tically increased cell death in 67% of cell lines and in 63% of primary samples. THE HOST GENETIC BACKGROUND MODULATES TREATMENT ACTIVI - TY AND TOXICITY IN FOLLICULAR LYMPHOMA: FIL-FOLL05 TRIAL Apoptosis, characterized by Annexin V staining, appearance of a subG1 peak 1 * 2 3 2 1 3 and caspase 9 activation, was accompanied by cell cycle arrest in G1 phase. G Palumbo , D Rossi , S Galimberti , A Bruscaggin , P Cava , E Ciabatti , A Dondi 4, F Angrilli 5, L Arcaini 6, G Bertoldero 7, F Merli 8, A Pulsoni 9, L Rigac - VD3 plus lenalidomide combination dramatically increased expression of the 10 11 12 13 14 15 2 BH3-only Bik without affecting expression of other Bcl2 molecules. By immuno - ci , G Rossi , C Rusconi , M Spina , C Stelitano , D Vallisa , G Gaidano , M Federico 4 precipitation assays, we showed that induced-Bik was not bind to anti-apoptot - 1 2 ic molecules Bcl2, BclxL or Mcl1 in treated cells but free to activate effectors Division of Hematology, AOU “Policlinico - V. Emanuele”, Catania, Division of molecules such as Bax. Moreover, silencing of BIK by siRNA prevented apop - Hematology, Amedeo Avogadro University of Eastern Piedmont, Novara, 3Dipartimento di Medicina Interna, Sezione di Ematologia, University of Pisa, tosis induced by VD3/lenalidomide, confirming the direct involvement of Bik in 4 cell death. Bik accumulation induced by lenalidomide and VD3 was not relat - Pisa, Department of Diagnostic, Clinical and Public Health Medicine, Univer - sity of Modena and Reggio Emilia, Modena, 5Department of Haematology, San - ed to an increase in transcription factor TEF expression but to an increase of 6 the ratio unmethylated over methylated of BIK CpG island. Similar epigenetic to Spirito Hospital, Pescara, Divisions of Hematology and Pathology, IRCCS Policlinico San Matteo, Pavia, 7Division of Oncology and Clinical Hematology, regulation of BIK expression was obtained with the inhibitor of methylation, 5- 8 azacytidine. Hospital of Mirano, Venice, Division of Hematology, S. Maria Nuova Hospital, Reggio Emilia, 9Department of Cellular Biotechnology and Hematology, La Summary / Conclusion: We show that lenalidomide and VD3, similarly to 5- 10 azacytidine, induce the expression of Bik via the unmethylation of BIK GpG Sapienza University, Rome, SOD Ematologia, Azienda Ospedaliero Univer - sitaria Careggi, Florence, 11 Division of Hematology, Spedali Civili of Brescia, island, which is responsible for cell death. Indeed, our results show for the first 12 13 time an original and non-toxic approach to significantly potentiate apoptosis Brescia, Division of Hematology, Niguarda Hospital, Milan, Division of Med - ical Oncology A, National Cancer Institute, Aviano, 14 Division of Haematology, induced by lenalidomide in MCL cells by simply adding physiological doses of 15 VD3. These data underline the interest 1) to measure the level of VD3 in MCL Ospedale “Bianchi-Melacrino-Morelli”, Reggio Calabria, Division of Medicine patients especially those receiving lenalidomide, 2) to define if this level is cor - and Hematology, G. da Saliceto Hospital, Piacenza, Italy related with the response, 3) to define whether supplementation with VD3 could increase response rate of patients receiving lenalidomide. Background: Follicular lymphoma (FL) is the most frequent indolent lymphoma subtype. Though most FL biomarkers rely on features of the tumor, the genet - ic background of the host may also be relevant for outcome. P287 Aims: Here we aimed at verifying the contribution of single nucleotide polymor - phisms (SNPs) to the prognostic stratification of FL patients treated with CXCR4 IS CRITICAL FOR NON HODGKIN LYMPHOMA CELL SURVIVAL, immunochemotherapy. INTERACTION WITH BONE MARROW MICROENVIRONMENT AND DRUG Methods: The study was based on 428/504 (85%) FL patients enrolled in the RESISTANCE IN VITRO AND IN VIVO IN ANIMAL MODEL FOLL05 phase-III prospective trial comparing R-CVP vs R-CHOP vs R-FM as K Beider 1,* E Rosenberg 1, M Abraham 2, I Weiss 2, H Wald 2, E Ribakovsky 1, initial treatment. Seventy-six patients were not assessable due to lack of bio - M Koren-Michowitz 1, A Avigdor 1, A Shimoni 1, A Peled 2, A Nagler 1 logical material. Candidate SNPs were selected because known to be relevant 1Bone Marrow Transplantation, Sheba Medical Center, Tel-Hashomer, 2Institute for: i) immunochemotherapy outcome or toxicity (MLH1 rs1799977, GSTA1 of Gene Therapy , Hadassah and Hebrew University, Jerusalem, Israel rs3957357, CYBA rs4673, NCF4 rs1883112, FCGR2A rs18011274, FCGR3A rs396991); ii) FL course (6p21.33 rs6457327). SNPs were genotyped by Sanger Background: CXCR4/CXCL12 chemokine axis has been implicated in the pro - sequencing or real time PCR on peripheral blood DNA samples. The primary gression of hematological malignancies. CXCR4 is highly expressed in a vari - endpoint was time to treatment failure (TTF). Median follow up of alive patients ety of B cell neoplasms, including NHL. CXCR4 inhibition in combination with was 34 months. rituximab has been shown to be an effective strategy in targeting lymphoma Results: Patients (n=428) were representative of the entire FOLL05 study cells in BM environment in disseminated NHL xenograft model. cohort and SNP genotypes distributed in Hardy-Weinberg equilibrium. Though Aims: To provide a mechanistic insight into CXCR4-regulated lymphomagen - FCGR2A and FCGR3A SNPs have been suggested to influence rituximab sin - esis in vitro and in vivo . gle agent activity in FL, our data document that they do not affect treatment Results: High mRNA and surface CXCR4 levels were detected in most of results when rituximab is combined with chemotherapy. Indeed, by pooled NHL cell lines (n=7) and primary samples from NHL patients with BM involve - analysis of the treatment arms, the 3-year TTF did not differ according to ment (n=7). Cell lines expressing high CXCR4 (BL-2 and Raji) demonstrat - FCGR2A (AA: 59% vs AG: 57% vs GG: 62%; P=.742) or FCGR3A (TT: 61% vs ed higher migration rate in response to CXCL12 and firmer adhesion to GT: 55% vs GG: 61%: P=.252) genotypes. These results were consistent also fibronectin and BMSC monolayer than low CXCR4-expressing BJAB cells. after compensating for treatment received and FLIPI by multivariate analysis Interaction with BMSC protected the cells with high CXCR4 from rituximab- (FCGR2A: P=.793; FCGR3A: P=.490). MLH1 is a component of the DNA mis - induced apoptosis. In contrast, BJAB cells with low CXCR4 were not protect - match repair that regulates the genotoxic effects of doxorubicin and impairs R- ed by stroma. To further investigate the role of CXCR4 in NHL progression, CHOP performance in diffuse large B-cell lymphoma. Consistently, the MLH1 in vivo xenograft model was established. Immuno-compromised mice inocu - genotype affected the 3-year TTF in the R-CHOP arm (AA: 66% vs AG: 68% lated subcutaneously with BL-2 cells produced highly invasive local tumors vs GG: 30%; P=.011), but not in arms lacking doxorubicin (p for R-CVP=.298; with BM dissemination and succumbed to lymphoma. In contrast, injection of p for R-FM=.601). The impact of MLH1 on TTF was independent (P=.004) after low CXCR4 expressing BJAB cells resulted in local slow-growing tumor devel - adjusting for FLIPI in the multivariate analysis. The remaining SNPs (GSTA1, opment without BM involvement and long-term animal survival. Notably, BL- CYBA, NCF4, 6p21.33) had no significant effect on TTF. Concerning toxicity, 2 cells that arrived to the BM demonstrated higher levels of mRNA and sur - the FCGR2A genotype, which modulates rituximab binding to effector cells, face CXCR4, compared to the bulk tumor, therefore suggesting the role of correlated with G3-4 neutropenia (AA: 56% vs AG: 43% vs GG: 40%; P=.026). CXCR4 in clonal selection and homing of lymphoma cells to the BM. To fur - Also the genotype of GSTA1, which is involved in cyclophosphamide detoxifi - ther establish the role of CXCR4 in lymphoma progression, we blocked cation, affected G3-4 neutropenia risk (CC: 58% vs CT: 43% vs TT: 42%; endogenous CXCR4 expression in BL-2 and Raji cells, using anti-CXCR4 P=.018). shRNA construct. CXCR4 silencing resulted in significant decrease in cell Summary / Conclusion: Taken together, these data indicate that: i) MLH1 viability in vitro , from 92% to 2% in BL-2 cells, and from 85% to 23% in Raji genotype is consistently associated with outcome in FL treated with R-CHOP, cells ( P< 0.002). Importantly, we found that anti-apoptotic proto-oncogene thus providing a more general and prospective validation of the usefulness of BCL-6 was down-regulated in the lymphoma cells following CXCR4 silenc - this host-related biomarker in R-CHOP treated lymphomas; ii) FCGR2A and ing. In accordance, CXCR4 silencing in BL-2 cells significantly ( P< 0.001) FCGR3A genotypes have no impact when FL is treated with rituximab combined inhibited local tumor growth and prevented NHL spread to BM. As comple - to chemotherapy; iii) GSTA1 and FCGR2A SNPs may represent biomarkers for mentary research strategy, we ectopically expressed CXCR4 in low-CXCR4 the identification of FL patients at risk of severe neutropenia. expressing BJAB cells. Exogenous expression of CXCR4 significantly increased the in vitro growth rate and promoted cell survival in the presence of BMSCs ( P< 0.01). Furthermore, interaction with BMSCs resulted in strong and prolonged pErk1/2 activation in exogenously expressing BJAB-CXCR4 cells, comparing to the native line expressing low CXCR4. Finally, CXCR4 over-expression significantly promoted the growth of xenograft subcutaneous tumors and their spread to the BM.

haematologica | 2013; 98(s1) | 123 18 th Congress of the European Hematology Association

P289 ER β agonists on lymphoma growth by grafting mice with lymphoma cells. RT- qPCR, microarray analysis and immunohistochemistry/immunofluorescence GENERATION AND CHARACTERIZATION OF A CD30 POSITIVE T-CELL were used in our study. LYMPHOMA MOUSE MODEL RESEMBLING HUMAN ANAPLASTIC LARGE Results: Treating lymphoma cells with estradiol or ERa selective agonist had CELL LYMPHOMA (ALCL) 1 * 1 2 2 3 1 minor or no effect on cell growth, while selective ER β agonist treatment showed C Klingeberg , A Illert , N Schneider , C Peschel , C Miething , J Duyster an anti-proliferative effect. When grafting mice with murine T lymphoma cells, 1First medical department, Universitätsklinikum Freiburg, Freiburg, 2Third med - 3 male mice developed larger tumors compared to female mice, a difference that ical department, TU München, München, Germany, Memorial Sloan-Kettering was abolished following ovariectomy, demonstrating estrogen dependent Cancer Center, New York, United States growth in vivo . To investigate whether lymphoma growth may be inhibited in vivo by ER β agonist treatment, mice were grafted with murine T and human B lym - Background: Anaplastic large cell lymphomas (ALCL) are a subgroup of aggres - phoma cells and treated with ERb selective agonists. Results showed that sive Non-Hodgkin-Lymphomas mainly affecting children and young adults. In treatment with ERb selective agonists strongly inhibited lymphoma growth of 60% of systemic ALCLs, a translocation t(2;5) (p23;q35) resulting in NPM-ALK several lymphoma types due to reduced proliferation and in some cases fusion gene expression is found. The constitutively activation of ALK tyrosine increased apoptosis. In addition, ERb selective agonists reduced lymphoma kinase expressed from the NPM-promoter causes increased proliferation and dissemination. Gene expression studies have identified target genes and mech - inhibition of apoptosis thereby promoting cell survival and tumorigenesis. anism that could explain the above effects of ERb agonists. Preliminary results Aims: Immunphenotypic characterization of human ALCLs revealed highly from immunohistochemical staining of primary mantle cell lymphoma material CD30-positive cells of T- or Null-Cell-origin and resulted in promising clinical showed that ERb is expressed in the tumor cells. trials with CD30-coupled antibodies. However, the impact of CD30 on diseases Summary / Conclusion: In summary, our results demonstrate an ER β ligand- development as well as NPM-ALK signal transduction in course of disease dependent antiproliferative effect on lymphoma cells expressing endogenous remain unclear and appropriate mouse models to answer these questions are ER β and that lymphoma cell growth and dissemination in vivo can efficiently missing. be reduced by ER β agonists. The results suggest that ER β agonists may be Results: In this regard, we established a retroviral murine bone marrow trans - useful in the treatment of lymphomas. The results may also explain the gen - plantation model resembling a human ALCL-like T-cell neoplasia. Therefore we der difference in incidence and prognosis of lymphomas. use an inducible Cre/loxP system where NPM-ALK expression is controlled and expressed in a special type of early T-cells. For generation of this vector, we inserted a floxed translational ‘stop-cassette’ between the retroviral promoter P291 MSCV-LTR and the NPM-ALK cDNA, which guaranties specific expression of NPM-ALK only in cells, where the enzyme Cre-recombinase is expressed. A COMPARATIVE ANALYSIS OF NEXT-GENERATION SEQUENCING AND Recognition of loxP-sites by Cre-recombinase leads in our system to deletion REAL-TIME QUANTITATIVE PCR FOR MINIMAL RESIDUAL DISEASE of the stop-cassette and consequently NPM-ALK expression. Using different DETECTION IN FOLLICULAR LYMPHOMA Cre-expressing cell types allowed us to study pathogenesis of ALCL in more C Pott 1,* L Monitillo 2, E Genuardi 2, B Mantoan 2, H Trautmann 1, M Kneba 1, detail. In our recent study, we infected bone marrow of transgenic mice express - M Brüggemann 1, M Faham 3, M Ladetto 2 ing Cre-recombinase under the control of the Lck-promotor with our MSCV- 1Second Medical Department, UNIVERSITY HOSPITAL SCHLESWIG-HOL - Stop-NPM-ALK-IRES-EGFP (MSNAIE) vector and transplanted it into lethally STEIN, CAMPUS KIEL, Kiel, Germany, 2Department of Molecular Biotechnol - irradiated C57/Bl6 recipient mice. With a latency of 4-5 months, these mice ogy and Health Science, University of Torino, Torino, Italy, 3Sequenta Inc, 400 developed Thy1.2-positive lymphomas and died from neoplastic infiltration of East Jamie Court, Suite 301 , San Francisco, United States bone marrow and lymphatic organs with T-cells. Immunphenotypic analyses confirmed T-Cell origin of the lymphomas and showed importantly highly CD30- Background: The detection of minimal residual disease (MRD) by t(14;18) expression. Staining of the different T-cell-subpopulations demonstrated high - Real-Time Quantitative (RQ) PCR has become an important tool for treatment est NMP-ALK expression in immature CD4/CD8 double negative T-cells and monitoring in follicular lymphoma (FL). However, only 50% to 65% of patients not fully differentiated CD4/CD8 double positive T-cells. Interestingly, FACS- can be assessed by t(14;18) RQ-PCR and alternative targets as the staining of the proliferation marker Ki-67 revealed highest expression in immunoglobulin heavy chain variable region (IGH) can be used only with lim - CD4/CD8 double negative T-Cell, in contrast to the other subpopulations where itations. IGH-based next-generation sequencing (NGS) might provide an alter - Ki-67 is less detected. Therefore we hypothesized, that the lymphoma initiat - native approach with further increase in sensitivity, specificity and accuracy. ing cell (LIC) must be within this early T-cell population. Most interestingly we Therefore we comparably analysed both approaches on diagnostic and post- found highest CD30-expression just in the same CD4/CD8 negative T-cell pop - treatment follow-up samples in 29 FL patients. ulation, pointing to a crucial role of CD30 in lymphoma initiation. To further sub - Aims: To verify the suitability of NGS for MRD detection we comparably stantiate our hypothesis we performed secondary and tertiary transplantations analysed diagnostic and post-treatment follow-up samples in 29 FL patients. with different sorted T-Cell subpopulation and indeed, the immature CD4/CD8 Methods: Overall, 206 samples (85 bone marrow, 114 peripheral blood, 6 stem double negative population was able to initiate lymphoma growth in recipient cell aliquots and one lymph node sample) were investigated from 29 FLenrolled mice. Future analyses of these mice will help to identify the leukemia initiating in clinical trials. Overall, 33 diagnostic and 173 follow-up (FU) samples were cell in ALCL. analysed. 23/29 patients had a PCR detectable t(14;18) rearrangement, 5 Summary / Conclusion: Taken together, our murine LckCre-NPM-ALK bone patients had a clonal IGH rearrangement only and one patient had no marker marrow transplantation model represents a precise and versatile tool to study by consensus PCR. RQ-PCR was carried out as previously published by our disease initiation and development resembling human ALCL. Moreover, the group and results were analysed according to ESHLO criteria. NGS was per - impact of specific proteins (e.g. CD30) in the course of disease can be formed independently at the Sequenta facilities in San Francisco and data addressed by combining Knockout (e.g. CD30)/LckCre transgenic mice with our remained blinded until comparison. Using universal primer sets IGH variable, model. diversity, and joining gene segments were amplified and sequenced with a coverage of 14 reads per each IGH molecule and analyzed using standardized algorithms for clonotype determination. Tumor-specific clonotypes were iden - P290 tified based on their high-frequency in diagnostic sample and then quantitated in FU samples. A quantitative and standardized measure of clone level among LYMPHOMAS AS ESTROGEN RELATED DISEASES - POSSIBLE TARGET all leukocytes was determined using internal reference DNA. Discordances of FOR ESTROGEN RECEPTOR BETA AGONIST TREATMENT. 1 * 1 1 2 3 4 MRD results by both methods were classified as follows: a positive/negative dis - K Yakimchuk , MS Hasni , J Guan , S Nilsson , S., M Jondal , B Sander , cordance between two results was defined as major when the positive result S Okret 1* 1 4 was >1 E-05 and minor when ≤1 E-05; a quantitative discordance was defined Dept. of Biosciences and Nutrition and Dept. of Laboratory Medicine, Karolin - as two positive results with a quantitative discrepancy >1 log. ska Institutet, Novum, SE-141 83 Huddinge, Sweden, 2Karo Bio AB, Novum, 3 Results: 29 patients were evaluable with at least one method. 15 patients SE-141 57 Huddinge, Sweden and Dept. of Microbiology, Tumor and Cell were evaluable for MRD by RQ-PCR and NGS. Here, in 97 FU samples a sig - Biology (MTC), Karolinska Institutet, SE-171 77 Stockholm, Sweden nificant concordance between both MRD methods could be demonstrated (r 2=0.80) ( P< 0.0001). Of these samples, 44 were MRD positive and 45 were Background: Lymphomas are generally not considered as endocrine related MRD negative with both tools .Quantitative discordances occurred in 12/44 cancers. However, most lymphoid malignancies show a gender differences in MRD+ samples where in 7 samples MRD was higher and in 5 was lower by incidence and prognosis with males being more affected. Furthermore, epi - NGS. A major discordance occurred in 4 samples where RQ-PCR was posi - demiological data in females show an association between reproductive hor - tive and NGS was negative. A minor discordance was detectable in 4 samples monal factors and oral contraceptives with a significantly reduced risk for Non- where in 2 samples RQ-PCR was positive and NGS was negative, and in the Hodgkin lymphomas, indicating a protective role of estrogens. More recent other 2 samples the opposite was correct. Seven patients were only quantifi - studies have demonstrated estrogen receptor beta (ERb) to be the major ER able by RQ-PCR while NGS did not identify an index clone for sequencing. In expressed in normal and malignant cells of lymphoid origin. all but one diagnostic samples demonstrated low level lymphoma infiltration Aims: The aim of the study was to investigate whether estradiol and selective with MRD below 10 -3 . In one of these cases, IG kappa could be successfully ERa and ER β agonists may affect lymphoma growth in culture and in vivo. sequenced for MRD indicating that not only low level MRD but also somatic Methods: We have analyzed the effects of estradiol and selective ERa and mutation of IGH is a potential pitfall for MRD detection by NGS. In 5 t(14;18)

124 | haematologica | 2013; 98(s1) Stockholm, Sweden, June 13 – 16, 2013 negative cases with a clonal IGH rearrangement IGH-RQ-PCR resulted in non P293 quantifiable assays. Here, NGS detected MRD successfully, as well as in one further case where an unusual large t(14;18) rearrangement did not allow MRD MINIMAL RESIDUAL DISEASE (MRD) DETECTION BY NEXT-GENERATION quantification by RQ-PCR. SEQUENCING AND REAL-TIME QUANTITATIVE PCR: A METHODICAL Summary / Conclusion: NGS represents a feasible tool for MRD monitoring COMPARISON IN ALL, MCL AND MM M Ladetto 1,* M Brüggemann 2, L Monitillo 1, S Ferrero 1, F Pepin 3, D Drandi 1, that allows analysis of a larger group of FL patients. Our results show that lym - 1 4 1 3 2 3 phoma infiltration of diagnostic samples is critical for identification of the tumor- A Palumbo , R Passera , M Boccadoro , V Carlton , H Trautmann , M Faham , C Pott 2 specific clonotypes by NGS and that different MRD methods may complement 1 each other to allow MRD assessment for the majority of FL patients. Department of Molecular Biotechnology and Health Science, University of Torino, Torino, Italy, 2Second Medical Department, University Hospital Schleswig-Holstein, Campus Kiel, Kiel, Germany, 3Sequenta Inc (www.sequen - P292 tainc.com) 400 East Jamie Court, Suite 301 , South San Francisco, United States, 4Statistical Consultant, University of Torino, Torino, Italy MYD88 L265P MUTATION IN WALDENSTROM MACROGLOBULINEMIA: INCIDENCE AND FUNCTIONAL STUDY Background: Real-Time Quantitative (RQ)-PCR-based MRD detection using S Poulain 1,* C Roumier 2, A Decambron 1, C Herbaux 3, E Bertrand 4, A Ren - primers derived from the immunoglobulin heavy chain variable region (IGH) is neville 5, A Daudignon 1, S Tricot 6, O Nibourel 5, C Roche-Lestienne 7, B Ques - an established disease monitoring tool in ALL, MCL and MM. It is highly sensi - nel 3, P Duthilleul 8, C Preudhomme 5, X Leleu 9 tive and has been standardized in the context of international cooperative 1Service d’Hématologie Immunologie Cytogénétique, CH de Valenciennes, groups such as the European Scientific Foundation for Laboratory Hemato- Valenciennes, 2Laboratoire d’Hématologie, Centre de Biologie Pathologie, oncology (ESHLO). However it has some limitations, including marker identifi - CHRU de Lille, Lillle, 3Service des Maladies du Sang, CHRU de Lille, 4INSERM cation failure, and false negatives due to clonal evolution. U837, IRCL, 5Laboratoire d’Hématologie, Centre de Biologie Pathologie, CHRU Aims: To verify if IGH-based next-generation sequencing (NGS) might over - de Lille, Lille, 6Service d’Hématologie Clinique, CH de Valenciennes, Valen - come some of these limitations of RQ-PCR and further increase sensitivity, ciennes, 7Laboratoire de Cytogénétique, CHRU de Lille, 8Service d’Hématolo - specificity, accuracy and reproducibility, we have performed comparison of the gie Immunologie Cytogénétique, CH de Valenciennes, 9Service des Maladie du two methods on diagnostic (DG) and post-treatment follow-up (FU) samples on Sang, CHRU de Lille, Lille, France a panel of 55 pts. Methods: 378 samples (62 DG, 316 FU) were collected from 55 pts enrolled Background: Mutation of MYD88 gene has recently been identified in activat - in clinical trials (15 ALL, 30 MCL, 10 MM). IGH-based RQ-PCR was carried out ed B-cell like diffuse B-cell lymphoma, and enhanced JAK STAT and NF-kB sig - as previously described [Ladetto et al , BBMT 2000; Brüggemann et al , Blood nalling pathways. Whole exome sequencing study in Waldenstrom macroglob - 2006], according to the ESHLO criteria [van der Velden et al , Leukemia 2007], ulinemia (WM) suggested a high frequency of MYD88 L265P mutation in WM. at two experienced MRD laboratories. NGS was performed at the Sequenta Although the genetic background is not fully deciphered in WM, the role of NF- facilities. Using universal primer sets, we amplified IGH variable, diversity, and kB and JAK STAT pathways has been demonstrated in WM. joining gene segments from gDNA. Amplified products were sequenced to Aims: We aimed to analyze MYD88 mutation in exon 5, to characterize the clin - obtain a high degree of coverage (14 reads per each IGH molecule) and ana - ical significance of this genetic alteration in 67 WM and to study the effects of lyzed using standardized algorithms for clonotype determination. Tumor-spe - MYD88 inhibition in WM cell lines cific clonotypes were identified for each patient based on their high-frequency Methods: 67 patients (42 males, 25 females) diagnosed with WM were includ - in DG sample and then quantitated in FU samples. A quantitative and standard - ed in this study. Patients were untreated at time of BM collection. Clinical fea - ized measure of clone level among all leukocytes was determined using inter - tures, immunophenotypic markers using flow cytometry (Matutes score panel, nal reference DNA. NGS analysis was performed independently under blinded CD38, CD138, CD27, CD80), conventional cytogenetic, FISH and SNP array conditions. Comparability of results by RQ-PCR and NGS was assessed by data (n=46) were analysed. B cells from bone marrow and T cells from blood bivariate correlations between methods (software R 2.15.1 package irr). Dis - were isolated respectively using B cell isolation and Pan T isolation kit (Myl - cordances were classified as follows: a positive/negative discordance was tenyi Biotech). For DNA sequencing of exon 5 of MYD88, the exon 5 of MYD88 defined as major when the positive result was >1 E-05 and minor when ≤1 E- gene was amplified from genomic DNA by PCR.The purified PCR products 05; a quantitative discordance was defined as the presence of two positive were directly sequenced in both directions using BigDye ® Terminator Cycle results with a quantitative discrepancy >1 log. Sequencing Kit (Applied Biosystems, CA, USA) and analyzed on the Applied Results: 51 pts (93%) were evaluable with at least one tool (RQ-PCR 45, NGS Biosystems 3130xl Genetic Analyzer. BCWM1, MWCL1 (WM cell lines with 49), 43 (78%) with both and 4 (7%) with none. Disease-specific success rates MYD88 L265P mutation), MEC-1, RL, and MM.1S cell lines were used in this are shown in Table 1. Sequences identified with both tools were identical or study. Cells were treated with a MYD88 inhibitor (MYD inh) ant its control pep - nearly identical in 41 cases and unrelated in two. Overall, 330 samples (87,3%) tide (Peptide crtl). Viability and cell growth of treated cells were determined were evaluated with at least one tool (RQ-PCR 279, NGS 316) and 265 (70%) using the MTS assay. Apoptosis was quantitated using annexin V - propidium with both. In terms of MRD output, concordance was significant ( P< 0.001) and iodide staining, mitochondrial membrane potential and caspase activity analy - 214/265 (80,8%) samples had an optimal concordance (96 MRD neg and 118 sis using flow cytometry. MRD pos). Of these major discordances were 16 (6%); minor discordances Results: MYD88 L265P mutation (MYD mut ) was observed in 79% of patients, were 24 (9,1%); quantitative discordances were 11 (4,1%). In 2 ALL clonal evo - including homozygous mutation in two patients (3%). MYD88 mutation was not lution hampered straightforward MRD assessment. In one case IGH RQ-PCR identified in T lymphocytes isolated from 4 WM patients. We haven’t observed underestimated MRD while a second RQ-PCR marker (TCRD) overlapped any other mutation on exon 5. We then sought for other mechanisms of MYD88 NGS. In a second case NGS did not detect the tumor diagnostic clone due to gene alteration, such as copy number alteration (CNA) and copy neutral –loss loss of the complete IGHV at relapse whereas the preceding IGHDJ was pre - of heterozygosity (CN-LOH) at MYD88 . We found an CN-LOH at MYD88 served and detected by RQ-PCR. locus in solely one patient (2%), and haven’t identified any deletion at 3p22. On Summary / Conclusion: NGS is an effective tool for MRD monitoring in ALL, the contrary, we observed a gain on 3 at 3p22 locus (including MCL and MM. Good concordance was seen in the vast majority of cases. How - MYD88 gene) in 7/57 (12%) patients. Taking together, we identified alteration ever, disease-specific pitfalls (clonal evolution, somatic hypermutations, fre - of the MYD88 locus in 85% of patients with WM, by either gain-of-function quency of complete IGH rearrangements) have to be considered for both meth - mutation (79%) or CNA (12%). Interestingly, we found gain on chromosome 3 ods. Prospective comparative analysis of unselected cases is required to ver - more frequently in the MYD wild group than in the MYD mut group (P=0.02). Twen - ify the clinical impact of NGS-based MRD assessment. ty one percent of the patients with WM had no mutation of MYD (MYD wild ), and were characterized with a female predominance, a splenomegaly, gain of chro - Table 1. Rates of success of RQ-PCR abd NGS among ALL, MCL, an MM mosome 3 and CD27 expression. We did not observed difference in terms of by patient. survival according to the MYD88 mutation status. MYD88 mutation was not related to deletion 6q, gain of 4, deletion 11q, deletion 17p, deletion 13q14 in our study. Overall, 63% of WM had at least one additional genetic alteration in the NF-kB pathway in our cohort, but no significant difference was observed accord - ing to the MYD88 mutation status. Inhibition of MYD88 signalling induced cyto - toxicity and inhibited cell growth in cells expressing MYD88 L265P mutation. Inhibition of MYD88 homodimerization also significantly inhibited MYD88 sig - nalling in the MYD mut as compared to the MYD wild cell lines, as exemplified by the marked downregulation of IRAK4 and STAT3 phosphorylation, downstream targets of MYD88. Summary / Conclusion: Our results confirm a high frequency of MYD88 L265P mutation in WM that may become a useful biomarker for diagnostic in WM and may help better understand the physiopathogeny of WM. From a ther - apeutic standpoint, this data also suggested that direct targeting of MYD88 sig - nalling may provide a novel approach for the treatment of MW in the future.

haematologica | 2013; 98(s1) | 125 18 th Congress of the European Hematology Association

P294 Moreover, Nipa-deficiency leads to premature senescence in cultured primary MEFs. Ectopic reexpression of Nipa resulted vice versa in delayed senescence B-CELL REPERTOIRE ANALYSIS OF INDOLENT MCL WITH SPLENIC of knockout MEFs. Next, we sought to know, whether increased apoptosis in PRESENTATION : A DISTINCT ONTOGENY FROM CLASSICAL MCL? -/- 1 * 2 2 1 3 Nipa c-Myc-transduced MEFs is dependent on a functional p53-Axis. Inter - C Sarkozy , M Cheminant , M Boudgrha , J Lazarovic , S Le Gouill , F Berg - estingly, the effect of Nipa deficiency on c-Myc-mediated transformation was er 4, A Traverse-Glehen 4, L Baseggio 5, P Felman 5, P Talmant 6, A Le Bour - 3 7 8 9 9 10 totally abolished by p53-knockdown. We observed no differences in focus for - geois , A Moreau , S Lissandre , V Eclache , F Baran-Marsazk , F Jardin , mation ability or growth behaviour in Nipa -/- MEFs with inactivated p53, sug - J Brière 11 , R Houlgatte 12 , F Davi 2, C Thieblemont 1 1 2 gesting the importance of p53 in Nipa-induced cell death. Looking in more Hematology, Hôpital Saint Louis, hemato-biology, Hôpital de la Pitié detail on the c-myc-p53 axis we detected a substantial increase in Arf-p19 lev - Salpetrière, Paris, 3hematology, CHU de Nantes, Nantes, 4hemato-pathology, -/- 5 6 els in Nipa cells. Moreover, Nipa-knockdown in Zn-inducible-Arf-NIH/3T3 hemato-biology, Centre Hospitalier Lyon Sud, Pierre Benite, cytogenetic, cells lead to stabilization of Arf p19. To test the impact of these findings in a rel - 7hemato-pathology, CHU de Nantes, Nantes, 8hematology, CHU de tours, -/- 9 10 evant in vivo model we intercrossed Nipa animals with a transgene EµMyc- Tours, hemato-biology, Hopital Avicennes, Bobigny, hematology, Centre Strain. Nipa -/- EµMyc TG/wt animals developed lymphomas within a significantly Henri Becquerel, Rouen, 11 hemato-pathology, Hôpital Saint Louis, Paris, +/+ TG/wt 12 shorter latency than Nipa EµMyc animals. Furthermore, lymphomas of Unité Inserm U954, inserm, Nancy, France knockout animals were more aggressive. FACS- and biochemical-analyses showed no gross differences between Nipa -/- and wt lymphomas except high - Background: Mantle-cell lymphoma (MCL) is a heterogeneous but well- ly elevated Arf-p19 levels in Nipa -/- lymphomas, pointing to an important role defined lymphoid malignancy characterised by the presence of a translocation, of Nipa in Myc-p19-signalling. the t(11;14)(q13;q32), inducing a dysregulation of the cyclin D1 expression, a Summary / Conclusion: Taken together our results highlight the functional key protein for the cell cycle at the G1-S phase transition. MCL is character - importance of the Nipa-p53-axis in cell cycle regulation and suggest that dereg - ized by a rapid and pejorative clinical evolution. Recently, it has been recog - ulation of the protein provides a substantial contribution during the process of nized that some patients experience a particularly indolent course with a pro - tumorigenesis. longed survival without therapy. These patients generally present with splenomegaly, blood involvement and minimal lymphadenopathy. However, indolent cases with a splenic presentation (splenic iMCL) remain incomplete - P296 ly characterized, especially regarding the B-cell reprtoire. Aims: The aim of our study was to characterize the clinical and biological fea - INFLUENCE OF THE B-CELL RECEPTOR (BCR) SIGNALING PATHWAYS tures of splenic iMCL, including their IGHV-IGHD-IGHJ repertoire and mutation - ON CD20 LEVELS IN TUMOR CELLS AND ANTITUMOR ACTIVITY OF al status. ANTI-CD20 MONOCLONAL ANTIBODIES Methods: Genomic DNA of 20 patients with splenic iMCL extracted from tis - M Winiarska 1,* K Bojarczuk 1, M Wanczyk 1, M Dwojak 1, P Zapala 1, N Miazek 1, sue specimen (blood, bone marrow biopsy, spleen) was used to characterise M Siernicka 1, J Golab 1 the IGHV-IGHD-IGHJ gene repertoire, to analyse somatic hypermutation (SHM) 1Department of Immunology, The Medical University of Warsaw, Warsaw, features, and to look for common CDR3 motifs. Poland Results: Patients characteristics include a median age of 65 years (range : 48- 84), 5 females and 15 males, ECOG-PS ≤ 1 for all patients, MIPI score low in 2, Background: Anti-CD20 monoclonal antibodies (mAbs) are widely used in the intermediate in 8 and high in 8. Median leucocyte count was 14.4G/L. Anemia treatment of non-Hodgkin’s lymphomas (NHL) and chronic lymphocytic was present in 6 patients, thrombocytopenia in 12, LDH > UNL in 4. The medi - leukemia (CLL). Combining new agents with already used anti-CD20 mAbs an follow up was 83 months (6-213). At time of analysis, 11 patients were alive seems to be a reasonable approach to further improve current therapeutic without receiving polychimiotherapy. Immunophenotype was available in 19 options. It seems that signaling via the aberrantly activated B-cell receptor patients: Matutes score was ≤ 3 (0-1 n=12, 5 n=2, 3 n=2), CD5 was negative (BCR) plays a key role in the pathogenesis of B-cell tumors. Blocking BCR sig - in 4 patients, IgM (+/-IgD) expression was reported in 13 patients/13 (IgG in 0), naling complex network holds a great therapeutic potential in both NHL and κ and λ in 12 and 7 cases respectively. A monoclonal productive IGH rearrange - CLL. Several trials are currently being conducted to investigate the effects of ment was obtained for 16 patients. There was a preferential usage of IGHV4- combination of BCR- targeting agents with anti-CD20 mAbs. To improve these 34 (4/16 cases) and IGHV3-7 (3/16 cases) genes. Interestingly, IGHV1-2 and therapeutic approaches it is utterly important to decipher actual mechanisms IGHV3-21, the most frequent genes used in splenic marginal zone lymphoma of interactions between BCR-targeted therapies and anti-CD20 mAbs in estab - (MZL) and classical MCL respectively, were not (IGHV1-2) or seldom (IGHV3- lished in vitro models. 21, 1/16) represented. While the IGHD repertoire had no particularity, there was an over-representation of the IGHJ4 gene (9/16) and an absence of IGHJ5. The vast majority of cases (14/16) had undergone SHM; the mean value for per - centage of identity to germline IGHV sequences was 96%, and 10/16 sequences could be considered as truly mutated (<97% identity). The median CDR3 length was 13 (6-20) and no stereotypic CDR3 motif could be identified. Summary / Conclusion: Although this constitutes a small series, our results suggest that iMCL with splenic presentation might have a different IGH reper - toire than those of and classical MCL as well as splenic MZL. Further insight into the potential of splenic iMCL to form a distinct entity is currently being investigated by comparative genomic analyses.

P295 NIPA DEFICIENCY LEADS TO ACCELERATION OF THE LYMPHOMA DEVELOPMENT IN EUMYC MICE A Illert 1,* C Klingeberg 1, C Albers 2, S Morris 3, U Keller 4, J Duyster 1 1Department of Internal Medicine I, University of Freiburg, Freiburg, 2Depart - ment of Internal Medicine III, Technical University of Munich , Munich, Ger - many, 3Department of Pathology, St. Jude Children’s Research Hospital, Mem - phis, TN, United States, 4Technical University of Munich, Munich, Germany

Background: Timely degradation of proteins that control cell proliferation and apoptosis is an essential mechanism in keeping normal growth from turning into runaway malignancy. We previously reported the cloning of NIPA (Nuclear- Interaction-Partner-of-ALK) and characterized it as a F-Box-protein that defines an oscillating E3-ubiquitin-ligase. Aims: To study in vivo function of the G 2/M checkpoint NIPA in greater detail, we inactivated the gene encoding Nipa using a conditional-knockout strategy. Results: Nipa -/- animals are viable, but sterile due to a block of spermatogen - esis. Our studies demonstrate that loss of Nipa has no substantive effect on physiological cell cycle progression of primary MEFs indicating that this cell cycle checkpoint is inactive under optimal proliferation conditions. Interesting - ly, Nipa checkpoint control can be unmasked by oncogenic c-Myc-transforma - tion. Here we show significant differences in c-Myc-induced transformation: -/- Focus formation ability of c-Myc-infected Nipa MEFs was greatly reduced. Figure 1.

126 | haematologica | 2013; 98(s1) Stockholm, Sweden, June 13 – 16, 2013

Aims: The aim of this study is to elucidate the role of BCR signaling pathways in P298 the regulation of CD20 levels in tumor cells and antitumor activity of anti-CD20 mAbs. PROGNOSTIC IMPACT OF EZH2 EXPRESSION, H3K27 TRIMETHYLATION Methods: The project is realized fully in in vitro settings in the models of human AND DNA METHYLATION IN DIFFUSE LARGE B CELL LYMPHOMA S Lin 1,* C Tsai 2, S Chuang 3, W Chou 4 lymphoma cells as well as primary cells from patients with B-cell tumors. Cells 1 are pre-incubated for 48h with inhibitors of BCR signaling (SYK, BTK, PLC, PKC, Department of Internal Medicine, devision of oncology and hematology, 2Department of Anatomical Pathology, Far Eastern Memorial Hospital, New PI3K, AKT) and subsequently tested using flow cytometry for their contribution to 3 4 antitumor effect of anti-CD20 mAbs (Figure 1B). Membrane level of CD20 anti - Taipei City, Department of Pathology, Chi Mei Medical Center, Tainan, Depart - gen is assessed with FITC-conjugated anti-CD20 antibody staining (Figure1A), ment of Internal Medicine, devision of hematology, National Taiwan University total level of CD20 protein is assessed in Western blotting (Figure 1D). Tran - Hospital, Taipei City, Taiwan scription processes are monitored with quantitative real time PCR (qRT-PCR) (Figure 1C), ChIP and EMSA. Moreover, stably transduced lymphoma cells with Background: International Prognostic Index (IPI) score is used to predict the silenced or overexpressed proteins of interest are employed. prognosis in diffuse large B cell lymphoma (DLBCL) for around 20 years. How - Results: The results of our preliminary experiments show that blocking BCR net - ever, few molecular prognostic factors were proposed. Overexpression of work at many stages of the signaling cascade with specific chemical inhibitors or Enhancer of zeste homolog 2 (EZH2) and decreased histone 3-lysine 27 tri- selective shRNA-mediated silencing of SYK or BTK results in considerable down- methylation (H3K27me3) are associated with poor prognosis in many cancers. regulation of CD20 level as determined with flow cytometry. Moreover, a 48-hour Promoter CpG island hypermethylation of tumor-suppressor genes is a com - incubation with BCR inhibitors leads to a substantial impairment of antitumor activ - mon hallmark of all human cancers. 5-methylcytosine (5-mC) levels in cancer ity of anti-CD20 mAbs. Selected inhibitors of BCR signaling considerably decrease cells can reflect the DNA hypermethylation status and measurement of 5- CD20 protein level in total cellular lysates as analyzed using Western blotting. In hydroxymethylcytosine (5-hmC) levels may estimate the DNA demethylation Raji cells incubated with selected BCR inhibitors qRT-PCR shows a significant status in cancer cells. A unique EZH2 Tyr 641 somatic mutation was detected decrease in CD20 mRNA level. The preliminary results from EMSA and ChIP indi - in DLBCL and follicular lymphoma. However, the clinical implications of DNA cate that mRNA decrease is not dependent on CD20 promoter and activity of tran - methylation status, DNA hydroxymethylation, EZH2 expression, the extent of scription factors known to regulate CD20 expression. Further studies elucidating H3K27me3 and EZH2 Tyr 641 somatic mutation in DLBCL patients have not the exact mechanisms of the observed phenomena will be performed. been studied in a comprehensive or integrated way. Summary / Conclusion: Blocking BCR complex network on nearly every step Aims: We aim to see significant impact of DNA methylation, DNA hydrox - of signal initiation and propagation considerably down-regulates CD20 levels ymethylation, EZH2 expression levels and mutation status, and H3K27 trimethy - what might have extremely important consequences for the anti-cancer therapy lation on the clinical and biological presentation of DLBCL. that is based on the use of anti-CD20 mAbs. These studies should provide us with Methods: We enrolled the 110 DLBCL patients with complete remission (CR) extensive knowledge on the biology of CD20 protein and pathways involved in after standard chemotherapy. These patients included 45 consecutive patients CD20 regulation. In light of our recent experiments therapeutic combinations of in Far Eastern Memorial Hospital and 65 consecutive patients in Chi Mei Med - BCR inhibitors and anti-CD20 mAbs-based modalities should be rationally and ical Center (diagnosed between 2002 and 2009). The demographic data, treat - consciously introduced into clinic in optimized therapeutic schemes. We hypoth - ment regimens, and response of disease were reviewed retrospectively. esize that results of our experiments may lead to identification of the most ben - Immunohistochemistry (IHC) was used to examine 5-mC, 5-hmC, EZH2 expres - eficial therapeutic modalities that would improve the quality of life of patients suf - sion and the extent of H3K27me3 in formalin-fixed, paraffin-embedded biopsy fering from B-cells originating tumors. specimens of DLBCL. DNA extraction from the tissues was examined for EZH2 Tyr641 mutation. Statistical analysis was performed with the Stata statistical software (Small Stata, version 11.0, Stata Corp, College Station, TX). P297 Results: Statistical analysis was performed in 110 CR patients with Rituximab- CHOP (R-CHOP) and CHOP regimen. Totally we recruited 70 male (64%) and GENETIC VARIATION IN INTERFERON REGULATORY FACTOR 4 AND 40 female (36%) with a median age 57 years. Sixty-three percent of the patients INTERLEUKIN 10 GENES, AND THE RISK FOR DIFFUSE LARGE B-CELL had stage I/II disease. According to the IPI, 76% of the patients were classified LYMPHOMA as low/low-intermediate risk (IPI=0-2). Thirty patients received CHOP-like reg - I Kim 1,* M Kim 2, E Lee 1, S Kong 3 imen and eighty patients received R-CHOP-like regimen as first-line chemother - 1Department of laboratory medicine, Pusan National University Hospital, Pusan apy. The median observation time for overall survival (OS) in the 110 patients , 2Department of internal medicine, Yeungnam University College of Medicine, was 49 mo. The clinical parameter of IPI score was correlated with OS. None Daegu, 3Department of laboratory medicine, National Cancer Center of Korea, of 48 patients harbored EZH2 mutation at Tyr641. Low expression 5-mC tend - Goyang, Korea, Republic Of ed to have lower H3K27me3 expression (P=0.019). And low expression 5-mC tended to have lower 5-hmC expression (P=0.002). There was no obvious rela - Background: Immune alteration is a major risk factor for non-Hodgkin lymphoma tionship between the expression of EZH2 and degree of H3K27me3 in DLBCL (NHL), but the specific immune mechanisms responsible remain unresolved. In tumor cells. There was no significant prognostic impact in single epigenetic a large consortial study, it has been recently reported that an increased risk for marker. Then we subdivide the patients by combination H3K27me3 with 5- NHL, especially the major lymphoma hmC. In this new-classification analysis, the high H3K27me3/ low 5-hmC Aims: We provide a comprehensive analysis of IRF4 and IL10 polymorphisms patients were associated with longer OS in univariate (P=0.013) and multivari - in a case-control study and risk association with DLBCL in Koreans. ate analysis (P=0.017). Methods: The case-control series consisted of 192 de novo DLBCL treated at Summary / Conclusion: High H3K27me3/ low 5-hmC expression was a pos - five hospitals throughout Korea from August 2001 through August 2009 and 192 sible favorable prognostic factor in DLBCL patients with CR after standard individuals from the population with age and gender matched healthy volunteers. chemotherapy. The incidence of EZH2 mutation at Tyr641 in our cohort is much The DNA samples were genotyped using matrix-assisted laser desorption/ioniza - lower compared with western countries. Low expression 5-mC tended to have tion-time of flight mass spectrometry (Sequenom, Inc., San Diego, CA). We geno - lower H3K27me3 expression and low expression 5-mC tended to have lower typed 15 haplotype-tagging SNPs (htSNPs) of IF4 (rs1877175, rs1877179, 5-hmC in DLBCL tumor cells. Our study suggests epigenetic markers may be rs2001508, rs2666957, rs2671422, etc.) and 7 htSNPs of IL10 (rs1518111, an informative biomarker for prognosis prediction in DLBCL. rs1800871, rs1800872, rs1800890, rs3021094, rs3024490, rs3790622) genes, respectively. We determined if the allelic distribution of the nine SNPs was con - sistent with Hardy-Weinberg equilibrium using the 2 test. All the genotype fre - quencies were in accordance with Hardy-Weinberg equilibrium. The allele and genotype frequencies of these SNPs were compared between the cases and con - trols using the 2 test or Fisher’s exact test. Unconditional logistic regression was done to estimate the odds ratios (OR) and 95% confidence intervals (95% CI) of the individual SNPs. The haplotypes were reconstructed according to the geno - typing data and the linkage disequilibrium status of these nine SNPs. Results: We did not find significant associations between IRF4 htSNPs and the risk of DLBCL. However, the minor allele heterozygotes of the rs3021094 htSNP of IL10 showed an increased risk of DLBCL (adjusted odds ratio = 1.453, P=0.034). On 10-million permutation testing, this haplotype including rs3021094 variant allele was significantly associated with an increased risk of DLBCL (P=0.001). Summary / Conclusion: This study presents several novel aspects of the genet - ic susceptibility to develop DLBCL. Our data do not statistically support the asso - ciation between IRF4 htSNPs and risk for DLBCL. However, we demonstrate that elevated DLBCL risk is associated with the rs3021094 htSNP of IL10. Larg - er studies that will focus on the role of the rs3021094 htSNP of IL10 for develop - ing DLBCL are needed in the future. Figure 1.

haematologica | 2013; 98(s1) | 127 18 th Congress of the European Hematology Association

P299 P300 BMI1 EXERTS ITS ONCOGENIC EFFECTS VIA ENHANCEMENT OF ANTI- THE ROLE OF CXC–CHEMOKINES IL-6, IL-8 AND CXCR2 RECEPTOR IN APOPTOTIC POTENTIAL IN MANTLE CELL LYMPHOMA LYMPHOPLASMACYTIC LYMPHOMA: CORRELATIONS WITH MICROVAS - K Teshima 1,* M Nara 1, A Watanabe 1, M Ito 1, S Ikeda 1, N Takahashi 1, K Oshi - CULAR CHARACTERISTICS AND CLINICAL FEATURES ma 2, M Seto 3, K Sawada 1, H Tagawa 1 G Levidou 1,* T Tzenou 2, M Kyrtsonis 2, E Nikolaou 2, N Kavantzas 1, D Mal - 1Department of Hematology, Nephrology and Rheumatology, Akita University tezas 2, K Xirokosta 2, E Koulieris 2, A Sepsa 1, K Bitsanis 2, I Pessach 2, Graduate School of Medicine, Akita, 2Department of Pathology, Kurume Uni - V Bartzis 2, M Dimou 2, P Panayiotidis 2, G Pangalis 2, E Patsouris 1, versity, Kurume, 3Department of Molecular Medicine, Aichi Cancer Center P Korkolopoulou 1 Research Institute, Nagoya, Japan 1Pathology, 2Haematology Section of the First Department of Propaedeutic Internal Medicine, University of Athens, Medical School, Athens, Greece Background: Mantle cell lymphoma (MCL) is categorized as an indolent CD5 + B cell lymphoma and is associated with numerous genomic copy number alter - Background: Increased neovascularisation is a vital process underlying the ations, including 9p21 deletion ( CDKN2A ) and 10p12 amplification ( BMI1 ). The development and progression of malignant neoplasms. Limited information on target gene of the 10p12 amplification is a proto-oncogene, BMI1 . Its overex - the subject is available for Waldenstrom’s macroglobulinaemia/Lymphoplasma - pression is recurrently observed in the blastoid variant of MCL, which suggests cytic lymphoma (WM/LPL), a rare and usually indolent B-cell lymphoma. BMI1 may play an important role in MCL tumorigenesis. Deletion of 9p21 is Aims: To evaluate the microvessel characteristics and IL-6, IL-8, CXCR2 and seen in 40-50% of MCL cases and results in dysregulation of the tumor sup - VEGF expression in the bone marrow of WM/LPL patients and investigate any pressor gene CDKN2A , which encodes p16 INK4a and p14 ARF . Earlier studies possible correlation with disease characteristics and outcome. have shown that BMI1 /Bmi1 exerts its oncogenic effects, at least in part, by Methods: Sixty-three patients were studied (47 WM and 16 LPL) for whom silencing the CDKN2A tumor suppressor locus, thereby promoting cell cycle paraffin-embedded tissue from bone marrow trephine biopsies performed at progression and suppressing apoptosis. Thus, Bmi1 has been thought to reg - diagnosis, before treatment was available. The microvascular characteristics ulate CDKN2A in MCL. However, previous report showed that MCL recurrent - were evaluated using CD34 stained slides. Microvessel density (MVD), total ly exhibited both homozygous deletion of 9p21 ( CDKN2A ) and amplification of vascular area (TVA) and several other size- and shape-related parameters 10p12 ( BMI1 ), suggesting that in that case, BMI1 regulates other tumor sup - were quantified in the region of most intense vascularization using a comput - pressors. erized image analysis software. Moreover, slides were immunostained with IL- Aims: The aim of this study is to determine the role of BMI1 in MCL, especial - 8, IL-6, CXCR2 and VEGF and evaluation was performed blindly using light ly in relapsed cases. And we identify a functional role of apoptotic regulation of microscopy. A Histo-score (H-score) based on the percentage of stained neo - BMI1 . plastic cells multiplied by staining intensity was calculated. Methods: We infected MCL cell lines with a robust inducible single-lentiviral Results: Thirty-two, 25 and 6 patients were classified into low, intermediate and vector knockdown reagent, pLKO-Tet-On, encoding either a non-targeting con - high risk respectively, according to the IPSSWM staging system. Seventy-six trol siRNA or a Bmi1-targeting siRNA (siBMI1). Then using puromycin selec - percent of the patients required treatment while 24% were asymptomatic and tion, stable polyclonal lines were generated. Chip assays were performed by were regularly followed. Median patients’ follow-up was 77 months. Microvas - use of SimpleCHIP TM Enzymatic Chromatin IP Kit (Cell Signaling Technology) cular characteristics, i.e microvessel density (MVD), total vascular area (TVA) according to manufacturer’s protocol. and several other size- and shape-related parameters, were evaluated in CD34 Results: We first examined BMI1 expression in primary cases and found that stained bone marrow slides using computerized image analysis. A Histo-score its expression was significantly higher at recurrence than at initial diagnosis. (H-score) based on the percentage of immunopositive neoplastic cells multi - Next, we found that transfection of siBMI1 induced apoptosis despite the plied by staining intensity was calculated for IL-6, IL-8, CXCR2 and VEGF absence of CDKN2A . Chip assay showed that Bmi1 interacted with pro-apop - immunohistochemical expression. MVD ranged from 7 to 393 µm 2 (median totic genes ( BCL2L11 /Bim and PMAIP1 /Noxa), which were recently shown to 36,5) and TVA from 2681 to 102480 µm 2 (median 32427). IL-6, IL-8, CXCR2 be Bmi1 targets. We also found that Bcl2 was up-regulated in MCL.Finally, we and VEGF expression was observed in 84%, 43%, 89% and 90% respective - found that bortezomib, which is known to be a proteasome inhibitor, induced ly. A positive correlation between IL-6 and CXCR2 H-scores was observed; IL- apoptosis with down-regulation of Bmi1 and up-regulation of Bim and Noxa in 6 and VEGF H-scores correlated with hypoalbulinemia and increased serum MCL. β2-microglobulin respectively and both with bone marrow lymphoplasmacytic Summary / Conclusion: We for the first time showed that BMI1 /Bmi1 is asso - infiltration and MVD. The degree of angiogenesis, microvessel shape, VEGF ciated with disease progression in MCL. We found that Bmi1 negatively regu - expression and CXCR2 expression correlated with a shorter time to first treat - lates BCL2L11 /Bim and PMAIP1 /Noxa to exert an anti-apoptotic effects in MCL ment in multivariate analysis. cells, which carry homozygous 9p21 ( CDKN2A ) deletions. Summary / Conclusion: In WM/LPL, increased expression of cytokines impli - cated in neovascularization and microvessel augmentation confer a more aggressive disease, reflected by an earlier need of treatment.

128 | haematologica | 2013; 98(s1)