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260 Homeobox NKX2-3 Is Over-Expressed in Human B-Cell Lymphomas and Drives Marginal Zone B-Cell Lymphomagenesis in Mice

Program: Oral and Poster Abstracts
Type: Oral
Session: 622. Non-Hodgkin Lymphoma - Biology, excluding Therapy: Molecular Mechanisms and Profiling
Monday, December 12, 2011: 7:00 AM
Room 33 (San Diego Convention Center)

Eloy F Robles, PhD1*, Beatriz Aldaz, MSc1*, Takashi Akasaka, MD, PhD2, Laura Macri Pellizzeri, MSc1*, Eduardo Martinez-Anso, PhD3*, Xavier Aguirre, PhD1*, Felipe Prosper, MD1,4, Idoia Martin-Guerrero, PhD5*, Victor Segura, PhD6*, Maria José Calasanz, PhD7*, Isidro Sanchez-Garcia, MD8*, Elena Campos-Sanchez, MSc9*, Cesar Cobaleda, PhD9*, Charlotte Cresson, MSc10*, Cyril Broccardo, PhD11*, Reiner Siebert, Prof., Dr. med.12*, Xabier Sagaert, MD13*, Martin JS Dyer, MA, DPhil, FRCP, FRCPath14* and Jose A Martinez-Climent, MD, PhD15

1Division of Oncology, Center for Applied Medical Research, University of Navarra, Pamplona, Spain
2Leicester University, MRC Toxicology Unit, Leicester, United Kingdom
3Hepatology and Gene Therapy, Center for Applied Medical Research, University of Navarra, Pamplona, Spain
4Hematology Service, Clínica Universidad de Navarra, University of Navarra, Pamplona, Spain
5Genetics, Institute of Human Genetics, University Kiel, Universitätsklinikum Schleswig-Holstein, Kiel, Germany
6Bioinformatics, Center for Applied Medical Research, University of Navarra, Pamplona, Spain
7Department of Genetics, University of Navarra, Pamplona, Spain
8Experimental Therapeutics and Translational Oncology Program, Instituto de Biología Molecular y Celular del Cáncer, Universidad de Salamanca, Spain
9Centro de Biologia Molecular Severo Ochoa, CSIC/Universidad Autonoma, Madrid, Spain
10Institut National de la Santé et de la Recherche Médicale, INSERM U1037, CHU Purpan, CPTP BatB, Toulousse, France
11Institut National de la Santé et de la Recherche Médicale, INSERM U1037, CHU Purpan, CPTP BatB, Toulouse, France
12Universitätsklinikum Schleswig-Holstein, Institute of Human Genetics, University Kiel, Kiel, Germany
13Department of Morphology and Molecular Pathology, University of Leuven, Leuven, Belgium
14MRC Toxicology Unit, Leicester University, Leicester, United Kingdom
15Division of Oncology, Center for Applied Medical Research CIMA, University of Navarra, Pamplona, Spain

While the molecular study of the common immunoglobulin (IG) translocations, hallmarks of B-cell lymphomas, led to the discovery of seminal cancer genes such as MYC and BCL2, cloning of other less frequent rearrangements has also identified genes with critical biological functions, including BCL10 and BCL11A. Therefore, molecular cloning of rare IG-related translocations may still pinpoint genes with unappreciated roles in lymphomagenesis. We identified a novel chromosomal translocation t(10;14)(q24;q32) involving the IGH locus in a case of mature B-cell lymphoma in leukemic phase. Molecular cloning by long-distance inverse PCR revealed involvement of NKX2-3. Subsequent screening of lymphoma cases with 10q chromosome breaks using fluorescence in situ hybridization identified a t(10;14)(q24;q12) translocation fusing NKX2-3 with TCRA. Both cases were classified as atypical low-grade mature B-cell lymphoma and exhibited increased expression of NKX2-3 with respect to normal B lymphocytes. In addition, NKX2-3 over-expression using quantitative RT-PCR and immunohistochemistry was detected in 42 of 166 (25%) primary mature B-cell lymphoma samples, including 15 of 29 (51%) splenic marginal zone lymphomas (SMZL), 14 of 46 (30%)  mucosa-associated lymphoid tissue (MALT) lymphomas, and 13 of 42 (31%) diffuse large B-cell lymphomas (DLBCLs), but not in follicular lymphomas (0 of 18), mantle cell lymphomas (0 of 8) or chronic B-cell lymphocytic leukemias (0 of 23). NKX2-3 belongs to the NKX family of homeodomain transcription factors that regulate cell-specific gene expression during differentiation and development. In mice, Nkx2-3 is essential for spleen and MALT development by regulating lymphocyte migration and homing to these sites. To determine whether NKX2-3 might, like some other family members, play an oncogenic role in hematopoietic neoplasms, Eμ-NKX2-3 transgenic mice were generated in which the EμSR enhancer drove restricted expression of human NKX2-3 to lymphocytes. Mice were fertile and developed normally. However, from 4 months of age, a progressive block in the pro-B (B220+CD19+Kit+) to pre-B cell (B220+CD25+) transition was detected in the bone marrow (BM), accompanied by a decrease in the number of circulating B220+IgM+ B lymphocytes. Notably, an expansion of CD21highCD23low marginal-zone splenic B cells was identified, which correlated with progressive spleen enlargement upon ultrasound monitoring of transgenic animals. From ~12 months of age, mice started to develop clinical signs of disease and were euthanized, showing massive splenomegaly (5-10 times larger than normal controls) in all cases (n=46). Histolopathological analysis of enlarged spleens revealed a complete red pulp infiltration of large and irregular nodules composed of cells with a biphasic morphology comprising an inner zone of small lymphocytes and a peripheral zone of larger lymphoid cells. Immunohistochemical studies showed that the infiltrating cells were mature CD20+IgM+IgD- B lymphocytes, with reactive CD3+ T lymphocytes, results that were concordant with flow cytometry studies. In 55% of the mice, additional extranodal tumors involving the lungs, liver and kidneys were detected, showing infiltrates of small mature B lymphocytes. Study of Igh, Igk and Igl rearrangements by PCR and sequencing revealed that most lymphomas were of clonal origin. Using gene expression microarray analysis, a significant overlap was found between the transcriptional signatures of the mouse NKX2-3 splenic lymphomas and human SMZLs, including genes known to be involved in human SMZL pathogenesis such as Notch2, Jun, Junb, Cyclin-D2, Ikzf3, Cxcr4, Traf5 and Maml2, as well as other genes implicated in mature B-cell lymphoma development such as Bcl3, Pax5, Bcl11a, Foxo3, Cebpb, Litaf, Socs1, IL10, Ccl5 and Cdkn1a. Taken together, these data indicate that the murine tumors closely resembled human splenic and extranodal marginal-zone (MALT) lymphomas. Furthermore, analysis of splenic and extranodal lymphomas from mice older than 18 months revealed areas of high-grade transformation to DLBCL, further highlighting the parallelism between splenic and human lymphomas. In conclusion, NKX2-3 protein is over-expressed in a subset of patients with SMZL, MALT lymphoma and DLBCL, and that the ectopic expression of NKX2-3 in mouse B lymphocytes recapitulates the main features of the human lymphoma counterparts.

Disclosures: Siebert: Abbott/Vysis : Speakers Honorarium.

*signifies non-member of ASH