Non-Hodgkin Lymphoma - Biology Analysed the Immunoglobulin (IG) Repertoire of 55 Cases of PIOL, the Largest Series to Date
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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 light chain rearrangements, were sequenced from PCR products obtained by amplification using either framework 1 or peptide leader primers. BMI1, THE POLYCOMB-GROUP GENE, 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 genes 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 Immunology 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 protein 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.