Transgenic mouse model of IgM+ lymphoproliferative disease mimicking Waldenström macroglobulinemia.

Waldenström macroglobulinemia (WM) is a low-grade incurable immunoglobulin M+ (IgM+) lymphoplasmacytic lymphoma for which a genetically engineered mouse model of de novo tumor development is lacking. On the basis of evidence that the pro-inflammatory cytokine, interleukin 6 (IL6), and the survival-enhancing oncoprotein, B cell leukemia 2 (BCL2), have critical roles in the natural history of WM, we hypothesized that the enforced expression of IL6 and BCL2 in mice unable to perform immunoglobulin class switch recombination may result in a lymphoproliferative disease that mimics WM. To evaluate this possibility, we generated compound transgenic BALB/c mice that harbored the human BCL2 and IL6 transgenes, EµSV-BCL2-22 and H2-Ld-hIL6, on the genetic background of activation-induced cytidine deaminase (AID) deficiency. We designated these mice BCL2+IL6+AID- and found that they developed-with full genetic penetrance (100% incidence) and suitably short latency (93 days median survival)-a severe IgM+ lymphoproliferative disorder that recapitulated important features of human WM. However, the BCL2+IL6+AID- model also exhibited shortcomings, such as low serum IgM levels and histopathological changes not seen in patients with WM, collectively indicating that further refinements of the model are required to achieve better correlations with disease characteristics of WM.

Non-Hodgkin lymphomas of mice.

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.

Inducible mutagenesis in TEPC 2372, a mouse plasmacytoma cell line that harbors the transgenic shuttle vector lambdaLIZ.

The plasmacytoma cell line, TEPC 2372, was derived from a malignant plasma cell tumor that developed in the peritoneal cavity of a BALB/c mouse that harbored the transgenic shuttle vector for the assessment of mutagenesis in vivo, lambdaLIZ. TEPC 2372 was found to display the typical features of a BALB/c plasmacytoma. It consisted of pleomorphic plasma cells that secreted a monoclonal immunoglobulin (IgG2b/lambda), was initially dependent on the presence of IL-6 to grow in cell culture, contained a hyperdiploid chromosome complement with a tendency to undergo tetraploidization, and harbored a constitutively active c-myc gene by virtue of a T(6;15) chromosomal translocation. TEPC 2372 was further characterized by the ability to respond to in vitro exposure with 4-NQO (4-nitroquinoline-1-oxide), an oxidative model mutagen, with a vigorous dose-dependent increase in mutagenesis that peaked at a 7.85-fold elevation of mutant rates in lambdaLIZ when compared to background mutant rates in untreated controls. Cotreatment with 4-NQO and BSO (buthionine sulfoximine), a glutathione-depleting compound that causes endogenous oxidative stress, resulted in a 9.03-fold increase in the mutant frequency in lambdaLIZ. These results demonstrated that TEPC 2372, the malignant plasma cell counterpart of the lambdaLIZ-based in vivo mutagenesis assay, may be useful as an in vitro reference point for the further elucidation of oxidative mutagenesis in lymphoid tissues.

Translocation remodeling in the primary BALB/c plasmacytoma TEPC 3610.

Myc-activating chromosomal 12;15 translocations, the hallmark mutations of inflammation-induced BALB/c plasmacytomas, have recently been shown to undergo remodeling by isotype switch-like genetic recombinations that remove approximately 180 kb of immunoglobulin heavy-chain sequence in the vicinity of the rearranged, expressed Myc gene. Here we combine cytogenetic data on the 12;15 translocation (SKY and FISH) with the molecular analysis of key junction sites (long-range PCR followed by DNA sequencing) to demonstrate that translocation remodeling occurred as an infrequent, stepwise, and disomic tumor progression event in the tetraploid, fully transformed, and transplantable plasmacytoma TEPC 3610. This result was used, in conjunction with previously obtained molecular data on five other primary plasmacytomas, to devise a hypothesis that predicts that the selective pressure to undergo translocation remodeling may be predetermined by the location of the break site in Myc. The pressure may be low if the break occurs 5' of the normal promoter region of Myc, but it may be considerably stronger if the break occurs 3' of the Myc promoter. Published 2001 Wiley-Liss, Inc.

Burkitt lymphoma in the mouse.

Chromosomal translocations juxtaposing the MYC protooncogene with regulatory sequences of immunoglobulin (Ig) H chain or kappa (Ig kappa) or lambda (Ig lambda) L chain genes and effecting deregulated expression of MYC are the hallmarks of human Burkitt lymphoma (BL). Here we report that lymphomas with striking similarities to BL develop in mice bearing a mutated human MYC gene controlled by a reconstructed Ig lambda locus encompassing all the elements required for establishment of locus control in vitro. Diffusely infiltrating lymphomas with a typical starry sky appearance occurred in multiple founders and an established line, indicating independence from positional effects. Monoclonal IgM(+)CD5(-)CD23(-) tumors developed from an initially polyclonal population of B cells. These results demonstrate that the phenotype of B lineage lymphomas induced by MYC dysregulation is highly dependent on cooperativity among the regulatory elements that govern expression of the protooncogene and provide a new system for studying the pathogenesis of BL.

Lymph nodes and Peyer's patches of IL-6 transgenic BALB/c mice harbor T(12;15) translocated plasma cells that contain illegitimate exchanges between the immunoglobulin heavy-chain mu locus and c-myc.

Hyperplastic plasmacytotic lymph nodes and Peyer's patches of 12 of 25 (48%) BALB/c mice that carried a human IL-6 transgene under the transcriptional control of the histocompatibility H-2L(D) promoter (BALB/c.IL-6 mice) were found to harbor 15 cell clones that contained in their T(12;15) translocation breakpoint regions illegitimate genetic recombinations between the upstream flank of the immunoglobulin heavy-chain C mu locus (5'-C mu) and c-myc (5'-C mu/c-myc+ clones). Similar 5'-C mu/c-myc+ clones were also detected in pristane-induced peritoneal granulomata (a significant source of IL-6 in situ) of three of 13 (13%) conventional BALB/c mice, but not in lymphoid tissues of pristane-treated BALB/c mice, nor in any tissue of untreated BALB/c mice. These findings provided strong evidence that IL-6 may be able to promote the growth and/or survival of clones that contained rearrangements between 5'-C mu and c-myc. Taken in conjunction with our previous observation that 5'-C mu/c-myc+ clones are the precursors for pristane-induced BALB/c plasmacytomas, the findings further suggested that IL-6 may play a pivotal role in the early stage of plasmacytoma development, by promoting tumor precursor cells. The BALB/c.IL-6 model of plasmacytomagenesis may be superior to the conventional BALA/c model because the putative plasmacytoma precursors appear to be more prevalent and in their development independent of treating the mice with inflammation-inducing plasmacytomagenic agents, such as pristane or silicone polymers.

Clonal diversification of primary BALB/c plasmacytomas harboring T(12;15) chromosomal translocations.

DNA sequence analysis of PCR amplified Igh/c-myc junction fragments of T(12;15) chromosome translocations and immunohistochemical determination of immunoglobulin isotype production were employed to study the clonal diversification of neoplastic translocated plasma cells that resided in peritoneal inflammatory granulomas of BALB/c mice harboring primary plasmacytomas. The diversity of plasma cells was found to take two major forms when the fine structure of the T(12;15) translocation was used as the clonotypic marker. First, mosaics of clones containing translocations that were apparently unrelated to each other were detected in nine out of 17 (53%) mice. Second, subclones derived from common T(12;15)+ progenitors by either secondary deletions in translocation breakpoint regions or aberrant isotype switching near translocation breaksites were found in five of 17 (29.5%) mice. When Ig expression was utilized as the clonotypic marker, clonal mosaics were shown to occur in all mice. This was demonstrated by the finding that the prevalent IgA- or IgG-producing plasmacytoma clone was invariably accompanied by smaller clones of IgG- or IgA-expressing neoplastic plasma cells, respectively. These results provided new insights into the clonal diversification at the terminal stage of plasmacytomagenesis. In addition, they suggested that BALB/c plasmacytomas may be uniquely useful for studying clonal diversity during B cell oncogenesis, since clonal evolution can be evaluated in a pool of tumor and tumor precursor cells that is clearly defined by the T(12;15) chromosomal translocation and the production of monoclonal immunoglobulin.

Cytogenetic analysis of the bipotential murine pre-B cell lymphoma, P388, and its derivative macrophage-like tumor, P388D1, using SKY and CGH.

Spectral karyotyping (SKY) and comparative genomic hybridization (CGH) were used to elucidate the divergent cytogenetic make-up of the prototypical bilineage lymphoblastic pre-B lymphoma, P388, and its progenitor macrophage-like tumor, P388D1. P388 was found to be diploid and genomically stable. P388D1 was triploid, highly unstable and characterized by numerous marker chromosomes (Chrs) and composite rearrangements. The karyotype of P388D1 was so complex that its clonal relatedness to P388 would have remained questionable without confirmation by molecular analysis of the clonotypic immunoglobulin heavy-chain and light-chain gene recombinations that coexisted in both tumors. The intrinsic instability of the P388D1 genome was indicated by the observation that only four out of 42 aberrations uncovered by SKY (in a total of 27 metaphases) occurred consistently (100% incidence), whereas 27 changes occurred non-randomly (27 to 96% incidence) and 11 alterations randomly (4 to 11% incidence). Persistent cytogenetic instability was also observed in P388 'macrophages' after phorbol ester- and ionomycin-induced conversion in vitro of P388 lymphoma cells. The 'cytogenetic noise' in these cells was manifested by a multiplicity of sporadic chromosomal aberrations; ie 25 distinct changes were identified by SKY in 40 metaphases. The results in P388D1 and P388 'macrophages' were interpreted to indicate that the myeloid differentiation program in the bipotential pre-B cell lymphoma P388 is invariably characterized by karyotypic instability. The study presented here demonstrates the power of the combined SKY and CGH approach to resolve complicated karyotypes of important and widely used mouse tumors.

Deletional remodeling of c-myc-deregulating chromosomal translocations.

Evidence is presented for the existence of a novel remodeling-by-deletion mechanism that alters the fine structure of c-myc-deregulating chromosomal translocations in t(12;15)-positive BALB/c plasmacytomas. DNA sequence analysis of the t(12;15) in five primary tumors revealed the co-existence of precursor cells harboring genetic recombinations between the immunoglobulin heavy-chain mu locus (Igh mu) and c-myc with clonally related progenitors containing rearrangements between the immunoglobulin heavy-chain alpha locus (Igh alpha) and c-myc. Clonal relatedness was based upon unique junction fragments between the switch region of Igh mu and c-myc. S mu/c-myc junctions are thus useful clonotypic markers for monitoring the conversion of Igh mu/c-myc-positive tumor precursor clones into Igh alpha/c-myc-positive plasmacytomas. Aberrant isotype switch recombination appears to be the most likely mechanism effecting this conversion event (other possibilities are discussed) which may help to explain the preferred usage of the Igh alpha locus in recombinations with c-myc in t(12;15)-positive plasma cell tumors in BALB/c mice.

Genetic recombinations between c-myc and Igh mu as precursors for recombinations between c-myc and Igh alpha in BALB/c plasmacytomas.

In this paper we present evidence that primary plasmacytomas harboring a balanced chromosomal translocation t(12;15) may consist of a mosaic of neoplastic clones of tumor cells, in which some of the clones are derived from common progenitors by large additional deletions which alter or "remodel" the fine structure of the t(12;15) on the c-myc-deregulating chromosome. This finding is based on the molecular analysis of the t(12;15) in three plasmacytomas, TEPC 1194, PCT 4127 and PCT 4132, in which primary clones of tumor cells with recombinations between the Ig heavy-chain (Igh) mu locus (Igh mu) and c-myc were found to be related to secondary clones of tumor cells with recombinations between the Igh alpha locus (Igh alpha) and c-myc. Clonal relatedness was based on unique junction fragments between S mu and c-myc that were found to be identical in Igh mu/c-myc and Igh alpha/c-myc breakpoint regions. S mu/c-myc breakpoints with adjoining sequences can thus be used as clonotypic markers, providing molecular fingerprints for the evolution of precursor cells with Igh mu/c-myc recombinations into progenitor cells with Igh alpha/c-myc recombinations. We propose that aberrant isotype switch recombination or illegitimate rearrangement in switch regions converts Igh mu/c-myc rearrangements, which are found very rarely in established plasmacytomas, into Igh alpha/c-myc rearrangements, which are present in about 80% of plasmacytomas with t(12;15). Remodeling of the t(12;15) results in closer juxtaposition of c-myc to the 3'-C alpha locus control region and probably in enhanced transcription of c-myc. We suggest that remodeling of c-myc-deregulating rearrangements constitutes a novel progression mechanism in malignant B-cell development.