Non-Hodgkin Lymphomas of Mice

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Blood Cells, Molecules, and Diseases (2001) 27(1) Jan/Feb: 217–222 doi:10.1006/bcmd.2000.0375, available online at http://www.idealibrary.com on

Non-Hodgkin Lymphomas of Mice Submitted 12/19/00 (Communicated by M. Lichtman, M.D., 12/19/00)

Mitsuo Hori,1 Shao Xiang,1 Chen-Feng Qi,1 Sisir K. Chattopadhyay,1 Torgny N. Fredrickson,1 Janet W. Hartley,1 Alexander L. Kovalchuk,2 Georg W. Bornkamm,3 Siegfried Janz,2 Neal G. Copeland,4 Nancy A. Jenkins,4 Jerrold M. Ward,5 and Herbert C. Morse, III1 ABSTRACT: Studies of lymphoid neoplasms occurring in normal or genetically engineered mice have revealed parallels and differences to non-Hodgkin lymphomas (NHL) of humans. Some mouse lymphomas have strong histologic similarities to the human NHL subsets including precursor B- and T-cell lymphoblastic, small lymphocytic, splenic marginal zone, and diffuse large-cell B-cell lymphomas (DLCL); whether molecular parallels also exist is under study. Others mouse types such as sIg⫹ lymphoblastic B-cell lymphoma have no histologic equivalent in human NHL even though they share molecular deregulation of BCL6 with human DLCL. Finally, Burkitt lymphoma does not appear to occur naturally in mice, but it can be induced with appropriately engineered transgenes. Key Words: BCL6; Burkitt lymphoma; mouse lymphoma; MYC; non-Hodgkin lymphoma.

NFS.V⫹ mice, ecotropic MuLV-negative NFS mice that are congenic for ecotropic MuLV induction loci of AKR and C58 and express ecotropic MuLV at high levels, develop B-cell NHL at high frequency (1, 6). Most AKXD recombinant inbred (RI) strains derived from crosses between high-virus AKR and low-virus DBA/2 also develop NHL at high frequency but with near equal representation of T- and B-cell types (7–9). Here we compare these mice for the incidence of NHL types, describe a novel model for mouse NHL, and relate the mouse lymphomas to those in humans.

INTRODUCTION Studies of naturally occurring mouse lymphomas have provided a wealth of information about the role of murine leukemia viruses (MuLV) in disease induction (1) and the histologic varieties found in different strains (2, 3). Other investigations have combined some of these understandings with analyses of mutagenic somatic integrations of MuLV, identifying common integration sites that affect expression of proto-oncogenes (4, 5). The genes affected by insertional mutagenesis in specific lymphoma types do not, in general, parallel those identified in related human non-Hodgkin lymphomas (NHL). For example, the Myc locus is frequently altered by MuLV integrations in mouse thymic Tcell lymphoblastic lymphomas (T-ALL) while altered expression of MYC in human T-ALL is rare, occurring in only 2% of cases.

MATERIALS AND METHODS Mice The origins of NFS.V⫹ and AKXD RI strains have been described previously (1, 6 –9). At au-

Correspondence and reprint requests to: Herbert C. Morse III, LIP, NIAID, 7 Center Drive, Room 7/304, MSC 0760, NIH, Bethesda, MD 20892-0760. Fax: (301) 402-0077. E-mail: [email protected]. 1 Laboratory of Immunopathology, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland 20892. 2 Laboratory of Genetics, Office of Laboratory Animal Science, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892. 3 GSF Forschungszentrum fu¨r Umwelt und Gesundheit, Institut fu¨r Klinische Molekularbiologie und Tumorgenetik, Munich, Germany. 4 Mouse Cancer Genetics Program, Office of Laboratory Animal Science, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892. 5 Veterinary and Tumor Pathology Section, Office of Laboratory Animal Science, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892. 1079-9796/01

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topsy, material from both sets of mice was saved for later preparation of DNA, flow cytometry, and histologic studies.

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TABLE 1 Spontaneous B-Cell Lineage Lymphomas of Micea Percentage of all lymphomas

Histopathology

Lymphomasb

Tissues were fixed in formalin (NFS.V⫹) or Fekete’s solution (AKXD), embedded in paraffin, sectioned, and stained with hematoxylin and eosin. All cases in both mouse sets were reviewed by one pathologist (TNF) to ensure consistency in diagnosis. Histologic diagnoses of AKXD cases were related to previously developed molecular studies of immunoglobulin and T-cell receptor gene organization, ecotropic proviral insertions, and alterations in the structure of common integration sites (7–9).

NFS.V⫹

AKXD RI

Molecular Studies

Precursor B cell Pre-B-ALL Mature B cell Small lymphocytic Follicular Splenic marginal zone Low-grade Intermediate High-grade Diffuse large cell Centroblastic Immunoblastic Lymphoblastic-like Histiocyte-rich All B lymphomas

DNA prepared from primary tumors or cell lines was examined by Southern blotting for the organization of Ig and TCR immune receptors and somatic integrations of ecotropic MuLV providing identification of cell lineage and clonality.

a NIAID/NCI classification system for spontaneous B-cell lineage lymphomas of mice. b Lymphomas of NFS.V⫹ mice (703 cases) and AKXD RI strains (376 cases) were classified according to combined histologic, phenotypic, and molecular criteria. The incidence of B-cell lineage tumor types among these lymphomas is indicated. c Numbers within brackets provide approximate values.

Flow Cytometry

[0]c

4

9 6

1 4

9 8 14

19 ⬍1 0

13 6 20 [3] 85

0 0 15 11 54

clude precursor B-cell lymphoblastic (pre-B ALL), small lymphocytic (SLL), follicular (FL), splenic marginal zone (MZL), and diffuse large cell lymphoma (DLCL) (Table 1) as well as plasmacytoma (PCT) (not shown and less than 1% of cases). The two sets of mice differed in the tumor types they experienced in several interesting respects including the much higher incidence of B-cell NHL in NFS.V⫹. First, AKXD mice were diagnosed with very few intermediate and highgrade MZL and no centroblastic or immunoblastic DLCL. Second, DLCL provisionally diagnosed as histiocyte rich were common among AKXD but not NFS.V⫹ mice. Finally, mice with more than one hematopoietic tumor, frequently two distinct types of NHL, comprised around 25% of cases in the AKXD series (9) but less than 5% in the NFS congenic series (9) (data not shown).

Single-cell suspensions prepared from spleen or lymph nodes of mice with lymphomas were stained with antibodies to cell surface antigens useful for distinguishing cell lineages and states of differentiation (6). RESULTS Comparisons of NFS.V⫹ and AKXD RI Strains for NHL Combined results of histopathologic, phenotypic, and molecular genetic studies of naturally occurring lymphomas in NFS.V⫹ and AKXD RI strains uncovered a spectrum of B-cell lineage lymphoma types (Table 1) as well as T-cell lymphomas of mostly thymic origin (not shown). These were diagnosed according to the criteria outlined in the proposed NIAID/NCI classification system for mouse hematopoietic neoplasms (1, 3, 6, 9), with a number of diagnostic categories appearing to have histologic parallels among human NHL. They in-

Surface Ig⫹ LL One diagnostic category of B-cell NHL with no clear histologic counterpart in humans is LL, comprising cells expressing Ig at the cell surface 218

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demonstrated that it occurred in the first intron of Bcl6, the most common site for BCL6 translocation in human DLCL (11). A number of additional cases with structural alterations in Bcl6 were found among AKXD LL, indicating that this change is not restricted to NFS.V⫹ lymphomas (9). It remains to be determined whether Bcl6 alterations in cases other than WEHI 231 can be ascribed to translocations. These findings serve to highlight the fact that cells with greatly different phenotypes can be associated with altered expression of the same oncogene. For example, translocations resulting in deregulated expression of CCND1 are common in human multiple myeloma, a disease of mature B cells, as well as in mantle cell lymphomas, tumors that arise from naı¨ve B cells (13). An alternative explanation might be that there are species-related differences in the way cells respond to activation of a proto-oncogene; Bcl6 activation results in mostly LL in mice and DLCL in humans. The issue of whether mouse sIg⫹ LL should be considered as a subtype of mouse DLCL clearly deserves consideration. Human DLCL have been found to exhibit point mutations in the first intron of BCL6 of either a translocated or germline allele (14). This observation may be explained by the fact that normal germinal center (GC) B cells also carry point mutations in the same region of the locus

TABLE 2 Lymphoblastic Lymphomas of Mice and Humans Subset

Human counterpoint

Genetics in mice

Precursor T cella Precursor B cellb Mature B cell

Yes Yes No

Myc, Nmyc1, Pim1 Evi3 Bcl6

a Precursor T-cell lymphoblastic lymphomas is a provisional designation for T-cell lymphomas of thymic origin that often have CD4, CD8, and TCR phenotypes of immature T cells. b Precursor B-cell lymphoblastic lymphomas are sIg⫺ counterparts of the human lymphoma type, usually with rearranged Ig heavy-chain but germline light-chain genes. sIg⫹ LL are usually IgM⫹ IgD⫺, CD5⫹. Genes found altered in naturally occurring mouse lymphomas of each type, primarily as a result of proviral insertional mutagenesis, are indicated.

(Table 2). In the WHO classification of human hematopoietic neoplasms (10), the term lymphoblastic is reserved for precursor T and precursor B lymphomas, and both of these lymphoma types also occur in mice. This leaves mouse tumors with LL cytology and sIg⫹ phenotype as a type unique to mice. To determine whether mouse sIg⫹ LL might relate molecularly to subtypes of human NHL in spite of their unique histologic niche, we studied the structure and expression of several oncogenes deregulated in human NHL, usually as the result of chromosomal translocations, initially in the NFS.V⫹ lymphomas. One of the genes evaluated was BCL6 (Table 3), a gene involved in chromosomal translocations in over 30% of human DLCL and approximately 15% of FL (11). We documented several cases with structural changes in the Bcl6 locus, one in a case of immunoblastic DLCL and others in LL (12); a structural change was also documented for the WEHI 231 cell line. Changes in the structure of proto-oncogenes in mouse lymphomas are almost always the result of mutagenic, somatically acquired MuLV proviral insertions, with a large number of different genes potentially involved in pathogenesis (5). The WEHI 231 cell line exhibited no new ecotropic MuLV insertions, suggesting another mechanism for the change in Bcl6. Using spectral karyotyping, we demonstrated that the cell line carried a balanced T(5;16) translocation, with Bcl6 mapping to the break point (12). Fine mapping of the breakpoint

TABLE 3 Comparison for Mouse and Human NHL for Characteristics of DLCLa Characteristic

Humans

Mice

Nodal or extranodal presentation Aggressive histologic appearance Varying cytology Centroblastic Immunoblastic Histiocyte- and/or T cell-rich Lymphoblastic lymphoma-like Characteristic mutation Translocation with multiple partners Ig-like hypermutation of BCL6 intron 1 In DLCL In normal germinal center B cells

⫹ ⫹

⫹ ⫹

⫹ ⫹ ⫹ ⫺ BCL6 ⫹

⫹ ⫹ (⫹) ⫹ Bcl6 ⫹

⫹ ⫹

⫺ ⫺

a

The terms histiocyte-rich, T cell-rich, and lymphoblastic lymphoma-like are provisional.

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(15). The mutations found are remarkably similar to those found in Ig variable regions, suggesting that the same mutational machinery is responsible for both (Table 3). A basis for reconsidering this conclusion comes from studies indicating that mouse GC B cells do not have mutations in the region of the Bcl6 first intron homologous to that shown to be highly mutated in human GC cells (16). In addition, a number of mouse NHL derived from post-GC B cells also lack point mutation in the first intron (M. Hori, manuscript in preparation). These studies suggest that the mutational machinery acting on Ig in human DLCL may actually be different from that affecting BCL6. Alternatively, species-specific differences may make the mouse Bcl6 sequences resistant to a common mutagenic mechanism.

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TABLE 4 Involvement of Proto-Oncogenes Complementing MYC for Transformation of B-Lineage Cells in Transgenic Mice (E␮-Myc, ␭-MYC) and Mice Injected with Pristane (PCT) Primary tumors and cell lines Tumor acceleration proviral tagging (E␮-MYC)

E␮-MYC

␭-MYC

PCT

Bmi1 Eis Gfi1 Pim1 Pim2

p53 p16 p19ARF Mdm2

p53 p16 p19ARF

p16

have been made using an Ig␬ LCR-like construct (H. C. Morse, unpublished data). Lymphomas appearing in mice bearing these transgenes have the histopathologic and cytologic features of human BL. Monoclonal tumors arising from an initial polyclonal population of B cells also exhibit a pattern of cell surface antigen expression similar to that seen in human BL. The p53–p16INK4A–p19ARF axis acts as a brake to the proliferative stimulus provided to cells by overexpression of MYC, resulting in cell cycle arrest or apoptosis (20). Approximately 30% of human BL exhibit mutations of p53, and another 30% or so exhibit deletions of p16 and p19 or methylation of the p16 promoter. Ongoing studies of mouse BL have shown that approximately 50% have mutations of p53 and that others appear to have deletions of p16/p19. It is important to note that p53 mutations are rare among other mouse lymphoid tumors, occurring at a comparable frequency only in LL of mice bearing an E␮-Myc transgene (Table 4) (21). High-level expression of Myc is not the only determinant of p53 mutation, however, as p53 mutations were found in less that 8% of mouse PCT bearing naturally occurring Ig/Myc translocations (22). It is noteworthy that an expression allele of p16 in susceptible BALB/c mice is also implicated in the development of PCT (23). Studies of E␮-Myc transgenic mice demonstrated that development of pre-B-cell lymphomas was greatly accelerated in mice infected with Moloney MuLV. A series of common proviral integration sites were identified, and flanking genes altered in expression as a result of these

Burkitt Lymphoma (BL) In humans, BL is a high-grade lymphoma with a striking starry sky appearance that invariably is associated with chromosomal translocations that juxtapose IgH or IgL loci with the MYC proto-oncogene, resulting in its activation. Comparable histologic findings were not observed in evaluations of more than 30,000 cases of lymphoma in NFS.V⫹ mice and 400 cases in AKXD mice. Structural alteration in Myc were observed in over 20 AKXD cases, but all were in T-cell lymphomas (9). Efforts to model BL in transgenic mice using Myc driven by IgH or IgL intronic enhancers were successful in inducing B-cell lineage tumors; most were pre-B lymphomas with some sIg⫹ B cells, but all had histologic features of LL instead of BL (17). It could thus be concluded that BL is not a naturally occurring NHL of mice and that induced overexpression of Myc in B cells was insufficient to induce a disease with features histologically similar to BL in humans. Recent experiments have shown that BL can be modeled in the mouse if Myc expression from a transgene is regulated in a similar manner to that in the human disease (18). The elements successfully employed to drive expression of a human MYC gene include elements from the 3⬘ end of the Ig␭ locus that function as a locus control region (LCR) in vitro (19). Somewhat similar findings 220

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insertions were characterized (Table 4) (4). There is no overlap between these genes and those mutated in non-accelerated Myc-induced diseases of mice. This suggests that the genes identified in virus-inoculated mice define a signaling pathway(s) functionally similar to that altered by mutation of the p53–p16 –p19 axis. The finding that Pim1 overexpression enhances the survival of hematopoietic cells is in keeping with this concept (24). A topic of major interest is raised by the differing features of human BL and mouse PCT, because both tumor types have translocations affecting MYC that would be expected to have highly similar if not identical effects on expression. Susceptibility to PCT induction is limited to a few strains, indicating prominent genetic effects. In addition, PCT arise in the inflammatory environment of mineral oil-induced peritoneal granulomas while BL, at least endemic cases, arise in gut-associated tissue. The toxic effects of oxygen and nitrogen radicals generated by activated peritoneal macrophages, an environmental milieu quite unlike that experienced by nascent BL cells, may elicit mutations that drive differentiation to plasma cells.

Mouse BL and mouse APL (25, 26) provide clear-cut demonstrations that detailed understanding of the molecular mechanisms involved in deregulated expression of proto-oncogenes in human hematopoietic malignancies can be used to manipulate the mouse by transgenesis to provide accurate models of human disorders. These models can be used to develop deeper understandings of disease pathogenesis, uncover new targets for treatment, serve as preclinical models for therapy, and provide rationales for preventative strategies. As one example, the BL model we describe is for the sporadic form of the disease where EBV is much less frequently involved (15 to 20%) than in endemic cases (95%). The model provides an opportunity to investigate the role of EBV by crossing our transgenic with those expressing EBNA or LMP genes. It will also clearly be of interest to ascertain whether the transgenic develops PCT when the gene is transferred to a PCTsusceptible BALB/c background or when the current B6 transgenic mice are treated with pristane. ACKNOWLEDGMENTS This work has been supported by grants to G.W.B. from Deutsche Forschungsgemeinschaft and Deutsche Krebshilfe. We thank Brenda Rae Marshall for excellent editorial assistance. This paper was presented at a Focused Workshop on Animal Models of Leukemia and Lymphoma jointly sponsored by the Leukemia & Lymphoma Society and the Mouse Models of Human Cancer Consortium of the National Cancer Institute on September 19 –20, 2000.

DISCUSSION These studies make three important points. First, they indicate that NHL of mice are considerably more heterogeneous than appreciated previously and suggest that thorough evaluations of genetic abnormalities in types with a histologic resemblance to human NHL are warranted. The opportunities to define molecular mechanisms of lymphomagenesis in high virus strains of mice has been greatly enhanced by the ability to use inverse PCR to rapidly define common integration sites (5). Second, they show that mouse and human NHL with common genetic alterations, such as Bcl6 translocations, may be histologically distinct. Finally, they demonstrate that lymphoma types not naturally seen in mice can be successfully modeled when molecular understanding of the primary genetic defects in human NHL is sufficiently advanced to permit construction of appropriate transgenes.

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