Anaplastic, Plasmablastic, and Plasmacytic Plasmacytomas of Mice: Relationships to Human Plasma Cell Neoplasms and Late-Stage Differentiation of Normal B Cells

Research Article

Chen-Feng Qi,1 Jeff X. Zhou,1 Chang Hoon Lee,1 Zohreh Naghashfar,1 Shao Xiang,1 Alexander L. Kovalchuk,2 Torgny N. Fredrickson,1 Janet W. Hartley,1 Derry C. Roopenian,3 Wendy F. Davidson,4 Siegfried Janz,2 and Herbert C. Morse III1

1Laboratory of Immunopathology, National Institute of Allergy and Infectious Diseases and 2Laboratory of Genetics, National Cancer Institute, NIH, Rockville, Maryland; 3Jackson Laboratory, Bar Harbor, Maine; and 4University of Maryland School of Medicine, Department of Microbiology and Immunology, Baltimore, Maryland

Abstract provide a perspective important to their classification and We have compared histologic features and gene expression nomenclature (1, 2). Diagnoses in both species are made through profiles of newly identified plasmacytomas from NFS.V+ a synthesis of histologic, immunohistochemical, and molecular congenic mice with plasmacytomas of IL6 transgenic, Fasl data, with the added contribution of clinical findings to analyses of mutant, and SJL-B2M / mice. NFS.V+ tumors comprised an human disorders. The histologic and molecular tools brought to overlapping morphologic spectrum of high-grade/anaplastic, bear on classifications of human lymphomas are frequently intermediate-grade/plasmablastic, and low-grade/plasma- reevaluated and revised in efforts to provide more precise cytic cases with similarities to subsets of human multiple diagnoses as guides in choosing among treatment options. myeloma and plasmacytoma. Microarray and immunohisto- One of the greatest challenges in developing consensus chemical analyses of genes expressed by the most prevalent nomenclatures of lymphomas for use by the clinical and scientific tumors, plasmablastic plasmacytomas, showed them to be communities is disease heterogeneity, perhaps best exemplified by most closely related to immunoblastic lymphomas, less so to diffuse large B-cell lymphoma. Hematopathologists have long plasmacytomas of Fasl mutant and SJL mice, and least to recognized the heterogeneity of this disease in humans, but efforts plasmacytic plasmacytomas of IL6 transgenic mice. Plasma- to define subgroups as distinct entities based on morphologic blastic tumors seemed to develop in an inflammatory envi- features have proven to be unsuccessful (3). The most common ronment associated with gene signatures of T cells, natural genetic marker—deregulated expression of BCL6 due to chromo- ¶ killer cells, and macrophages not seen with plasmacytic somal translocations or mutations of 5 regulatory sequences—can f plasmacytomas. Plasmablastic plasmacytomas from NFS.V+ be identified in only 50% of cases (reviewed in ref. 4). In addition, and SJL-B2M / mice did not have structural alterations in gene expression profiling, despite tremendous promise, has failed Myc or T(12;15) translocations and did not express Myc at to establish a comprehensive consensus molecular approach to high levels, regular features of transgenic and pristane- subset identification or outcome prediction (5, 6). induced plasmacytomas. These findings imply that, as for Multiple myeloma is another B-cell lineage disease entity, one human multiple myeloma, Myc-independent routes of trans- that is diagnosed histologically with relative ease but that has formation contribute to the pathogenesis of these tumors. recently been found to be unexpectedly heterogeneous in terms of These findings suggest that plasma cell neoplasms of mice and numerical and structural cytogenetic abnormalities, and gene humans exhibit similar degrees of complexity. Mouse plasma- expression profiles (reviewed in ref. 7). Recent studies suggest that cytomas, previously considered to be homogeneous, may thus this heterogeneity can be distilled to define eight subtypes of the be as diverse as their human counterparts with respect to onco- disease (8), raising the possibility that these represent eight disease genic mechanisms of plasma cell transformation. Selecting entities, each with its own therapeutic targets. Of interest, earlier specific types of mouse plasmacytomas that relate most histologic studies of large series of multiple myeloma cases also closely to subtypes of human multiple myeloma may provide identified as many as seven or eight histologic subtypes (9–11) with new opportunities for preclinical testing of drugs for treat- prognostic implications. Notably, an overlapping spectrum of ment of the human disease. [Cancer Res 2007;67(6):2439–47] histologic types has been described for patients with another plasma cell disease, extraosseus plasmacytomas (12), although, to Introduction our knowledge, no attempt was made to assess the prognostic potential of these subtypes. It is well established that different classes of mature B-cell Although mice rarely develop bone marrow plasma cell tumors neoplasms of humans and mice exhibit features that mimic specific with similarities to human multiple myeloma (13, 14), extraosseus stages of normal B-cell differentiation and that these similarities plasmacytomas develop spontaneously in some strains such as SJL/ J (15), are readily induced in others, including BALB/c (16) and NZB (17), and occur at variably high frequencies in a number of model Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/). systems. Published studies from our laboratories and others have Requests for reprints: Herbert C. Morse III, Laboratory of Immunopathology, revealed that these plasmacytomas are heterogeneous and can be National Institute of Allergy and Infectious Diseases, NIH, TwinbrookI, Room 1421, divided into subtypes based on histologic features and gene Rockville, MD 20852. Phone: 301-496-6379; Fax: 301-402-0077; E-mail: [email protected]. I2007 American Association for Cancer Research. expression profiles, some with human counterparts (18–23). Here, doi:10.1158/0008-5472.CAN-06-1561 we describe a new set of mouse plasmacytomas that developed in www.aacrjournals.org 2439 CancerRes 2007; 67: (6). March15, 2007

NFS.V+ mice (24). Histologically, they comprise three subsets—low missing value genes as predictors and a gene with a missing value as the grade/plasmacytic, intermediate grade/plasmablastic, and high response variable. Genes with missing values were added to the model one grade/anaplastic—that closely parallel the terminologies suggested at a time. This generated a matrix containing 11,181 genes without any by Bartl et al. (9) for variants of human multiple myeloma. We also missing values that was used for the final analyses. The raw intensity ratio was transformed into a logarithmic value log . Two-way hierarchical show that, in contrast to plasmacytomas from pristane-treated (16) 2 + clustering (Ward method) of genes against mouse lymphoma samples was or IL6 transgenic mice (20), NFS.V cases do not exhibit structural done using software developed at NIH.7 alterations in Myc and do not express Myc at high levels, indicating that other transforming pathways are in play. The possible Results relations of this heterogeneity to defined, distinct pathways of plasma cell differentiation, to subtypes of plasma cell neoplasms in Occurrence and histologic features of plasma cell neo- humans, and to tumor progression are discussed. plasms. Among f2,700 cases of hematopoietic tumors studied at necropsy in our laboratory over the last 5 years, 48 (1.8%) were Materials and Methods diagnosed as plasma cell–derived neoplasms that histologically seemed to be less mature than those of pristane-treated or Mice, histology, and immunohistochemistry. The characteristics of IL6 transgenic mice. All but three had splenic involvement, with NFS.V+ congenic (24) BALB/c-IL6 transgenic (20), SJL-h2M knockout (21), weights averaging f1.3 g (range 0.1–3.0 g); most had affected BALB/c-Fasl/Fasl mutant (18, 21), and n-MYC transgenic mice (25) were as previously described. Mouse protocols were approved by the Animal Care lymph nodes; and many exhibited infiltrates of liver, lung, or f and Use Committees of National Institute of Allergy and Infectious Diseases kidney. The average age at diagnosis was 450 days (range 116– (NIAID), National Cancer Institute (NCI), and the University of Maryland. At 788 days), and the male to female ratio was f1:1. Thirty-eight + necropsy, selected tissues were fixed in formalin for histologic studies and cases occurred in NFS.V mice that express ecotropic murine immunohistochemistry. Samples of spleen and/or lymph nodes were snap leukemia virus at high levels (24), three in virus-negative NFS frozen for later preparation of DNA and RNA. Histologic diagnoses were congenics, three in mice with Myc knocked into the IgH locus A made according to the Bethesda classification of mouse lymphoid (strain iMycE ; ref. 22), three in B6 mice bearing a n-MYC transgene neoplasms (2). Features of diffuse large B-cell centroblastic and immuno- (25), and one in a B6.me/+ mouse. blastic lymphomas have been described (2, 24). Immunohistochemical The neoplasms exhibited a broad morphologic gradation, studies were done using the panel of antibodies listed in Supplementary ranging from uniform populations of mature-looking cells, to Table S1 and procedures described previously (26). DNA analyses. High-molecular-weight DNA was digested, separated more immature forms, to anaplastic cells. The occurrence of these electrophoretically, and transferred using standard techniques (18, 20, 24). cells in large nodules or sheets and the presence of binucleate cells The membranes hybridized with 32P-labeled probe J11 and a Myc exon 2 differentiated them as neoplasms from accumulations of normal, probe for studies of immunoglobulin heavy chain and Myc gene reactive plasma cell. Similar morphologic variants of malignant organization, respectively. plasma cell have been described for human multiple myeloma and Interphase dual-color fluorescence in situ hybridization. Fluores- plasmacytomas. In addition, an association between plasma cell cence in situ hybridization (FISH) was done on formalin-fixed, paraffin- maturity and survival of patients with multiple myeloma has been 5 embedded sections as described. Bacterial artificial chromosome mapping recognized for >50 years (27). Subtypes defined by cytologic a to Myc located on chromosome 15D2-3 (D15Mit17) and to C constant features have numbered between two and seven in various studies region of IgH on chromosome 12F (189A22) were labeled using digoxygenin- (9–11, 27–31) but can be generalized to three: (a) low grade/well and biotin-nicktranslation kits(Roche, Indianapolis, IN), respectively, and visualized by avidin Alexafluor 568 (Molecular Probes, Invitrogen, Carlsbad, differentiated/plasmacytic; (b) intermediate grade/plasmablastic; CA) and sheep antidigoxigenin fluorescein Fab (Roche). Confocal Z-stack and (c) high grade/pleiomorphic/anaplastic. We adopted the images taken at 0.5-Am intervals were acquired on an IX81 fluorescent plasmacytic, plasmablastic, anaplastic system to categorize mouse microscope (Olympus Optical, Tokyo, Japan) with a 60 or 100 oil lens. plasmacytomas. Merged pseudocolor maximum-projection images were generated using The first two types of plasmacytoma, plasmacytic and plasma- Slidebooksoftware (Intelligent Imaging Innovations, Santa Monica, CA). blastic, are fairly well defined from a cytologic perspective. The Oligonucleotide microarrays and analysis. Microarray experiments plasmacytic type (Fig. 1A) consists mainly of mature or fairly f were done as described (21) using chips comprising 14,000 mouse gene mature plasma cells, usually arranged in solid sheets. The cells have targets represented by 70mer oligonucleotides (Compugen, Jamesburg, NJ) a low nuclear to cytoplasmic ratio, abundant basophilic cytoplasm, and printed by the NIAID Microarray Research Facility.6 Total RNA was often a large juxtanuclear hof (Golgi), and a clockface nucleus extracted from primary tissues. A reference sample was prepared by pooling equal amounts of RNA from a panel of cell lines (21). cDNAs were labeled with clumped chromatin. Cytoplasmic immunoglobulin can readily with Cy3 and Cy5 dyes for primary tissue and reference samples, be detected by PAS staining (sometimes revealing Mott cells or respectively, and hybridized to the chips. Data from the scanned chips Russell bodies; Supplementary Fig. S1A) or immunohistochemistry were stored at the microarray database maintained by the Center for (Supplementary Fig. S2). Binucleate cells and mitoses are Information Technology, NIH. sometimes seen, but rarely. This cell type is typical of the The microarray data set was organized and analyzed using SAS software pristane-induced plasmacytomas of BALB/c mice (16) and many (SAS Institute, Cary, NC). To remove variation among the hybridizations, all plasmacytomas that develop in IL6 transgenic mice (20). In our the hybridizations (chips) were scaled together using a linear procedure cases, the spleen and lymph nodes are usually affected, but based on a selected set of features by setting the summed abundance of the infiltration of the kidneys, liver, and lung may also occur. This type selected features equal to a constant (linear scaling). An imputation method must be differentiated from accumulations of plasma cell reflecting was developed based on the partial least square algorithm using SAS software. Briefly, a partial least square model was built using all the non– the effects of chronic inflammation on draining lymph nodes or of systemic inflammatory, infectious, or autoimmune disorders

5 http://www.riedlab.nci.nih.gov/protocols.asp#h3 6 http://madb.niaid.nih.gov 7 http://discover.nci.nih.gov

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Figure 1. Histologic features of immunoblastic lymphomas and plasmacytoma. A, plasmacytic plasmacytoma with almost all mature cells with central nucleoli in clock-face nuclei, basophilic cytoplasm, and discernible Golgi. A binucleate cell is near the center of the field, and somewhat less mature plasmablasts are near the top. B, plasmacytic plasmacytoma, more pleiomorphic with a greater proportion of plasmablasts than in (A). Some apoptotic bodies and a mitosis can be seen. C, plasmablastic plasmacytoma with a near-uniform population of plasmablasts that vary significantly in size and have unusually large, magenta nuclei. Several mitoses are present along with active apoptosis. Occasional mature cells are seen, and one immunoblast at the top. D, plasmablastic plasmacytoma with great variation in cell size, apoptosis, and mitosis. E, anaplastic plasmacytoma with a mixture of immunoblasts and anaplastic cells as well as some plasmablasts. Note the absence of mature cells. F, immunoblastic lymphoma with large magenta nucleoli attached to the nuclear membrane. Note the presence of centroblasts with nuclei featuring vesicular chromatin and two nucleoli sited on the nuclear membrane and the absence of plasmablasts. on both spleen and lymph nodes. In reactive lymph nodes, the nohistochemical studies demonstrating low to intermediate levels medullary cords are filled with mature plasma cell, and the cortex of cytoplasmic immunoglobulin provide important support is usually enlarged containing numerous, active germinal center (Supplementary Fig. S2). Mitoses, tingible body macrophages, and (GC). In spleens of mice with inflammatory conditions, the white apoptotic bodies are constant features, as is diffuse infiltration of pulp is expanded and displays multiple large GC. The red pulp spleen, lymph nodes, and other tissues. In about half the cases, features increased myeloid activity and accumulations of plasma there may be areas resembling the plasmablastic type, but the cell, often adjacent to the bridging channels between follicles and general aspect is one of a very undifferentiated population. the red pulp. The end-stage differentiation of plasma cell in lymph Theanaplasticcasescanbeverydifficulttodistinguish node medullae and the splenic red pulp is uniform, and mitoses are from immunoblastic lymphomas (Fig. 1F), as both contain blasts absent. In mice with localized inflammation, lymph nodes other with vesicular nuclei and prominent magenta nucleoli frequently than those draining the site are usually normal. fixed to one side of the nuclear membrane (Fig. 1E and F; The plasmablastic type is distinguished by aggregates of Supplementary Fig. S1B). Immunoblastic lymphomas often stain plasmacytoid cells in all distinguishable stages of plasma cell for cytoplasmic immunoglobulin (Supplementary Fig. S2), but fail differentiation, especially plasmablasts (Fig. 1B and C). This is to exhibit most other signs of progression toward terminal sometimes evident in single fields of spleen, lymph node, or liver differentiation such as accumulations of plasmablasts or expres- sections. Cytologic features of plasmablasts are quite distinctive, sion of surface CD138, cytoplasmic XBP1, or nuclear IRF4. featuring a thickened, sometimes irregular, nuclear membrane It is not uncommon to see morphologic differences among surrounding a vesicular nucleus with a single large, central nu- tumor cells present in spleen versus lymph nodes or among cleolus and plentiful violaceous cytoplasm. Unlike the plasmacytic different affected nodes in a single animal. The spectrum in any type, mitoses and active apoptosis are frequently seen. Areas of one case, however, is usually limited to overlaps of plasmablastic infiltration are irregular and may be widespread, differentiating with plasmacytic areas and anaplastic with plasmablastic areas. Of them from the more confined presentation of the plasmacytic type. interest, composite lymphomas were identified in the spleens of The presence of GC with a transition zone blending into cells with four cases, each comprising splenic marginal zone lymphoma and a an obvious plasmacytoid phenotype may be a prominent feature coexisting plasmablastic or anaplastic plasmacytomas. In humans, of the plasmablastic class, particularly in SJL disease. nodal MZL (NMZL) may sometimes exhibit prominent plasma cell The anaplastic form of plasmacytoid neoplasms presents a differentiation (32) and some nodal lymphomas originally diag- considerable diagnostic challenge. Morphologically indistinguish- nosed as plasmacytomas have been shown to have features of able large blast cells, often with folded nuclear membranes NMZL (33). and open vesicular nuclei, can be numerous (Fig. 1D and E; It has also been suggested that some nodal plasmacytomas with Supplementary Fig. S1B). Recognizable plasma cell at any stage of features of NMZL may arise from plasma cell variants of localized differentiation are rare. Consequently, reliance must be placed on Castleman’s disease, a nonneoplastic lymphoproliferative disorder the identification of plasmablasts and transitional forms between of humans (34). Recent studies showed that the plasma cell variant plasmablasts and immunoblasts. The presence of a heavily of multicentric Castleman’s disease is very responsive to treatment basophilic cytoplasm is sometimes very informative, and immu- with monoclonal anti–interleukin-6 receptor monoclonal antibody www.aacrjournals.org 2441 CancerRes 2007; 67: (6). March15, 2007

Figure 2. Immunohistochemical analyses of a plasmacytic plasmacytoma. Sections of formalin-fixed, paraffin-embedded tissue were stained with H&E (A)or reacted with antibodies to IRF4 (B), CD138 (C), and XBP1 (D), followed by appropriate second antibodies labeled with horseradish peroxidase. Original magnifications, 40.

(35) demonstrating, in parallel with our IL6 transgenic mice, the the frequency of positive tumor cells and their staining intensity. importance of this cytokine to premalignant expansion and The results of these studies (Table 1) showed that for each B-cell transformation of plasma cell. GC were shown to be the source marker, the frequency of positive cases and the expression levels for of IL6 in the human disorder. the positive cases decreased progressively with the transitions from Using the criteria described above to subset the 48 cases of centroblastic to immunoblastic lymphoma to anaplastic/plasma- plasmacytomas originally identified, 33 were classified as plasmablastic plasmacytomas to plasmacytic plasmacytomas. Conversely, blastic, 10 as anaplastic, and 5 as plasmacytic. SJL cases exhibited for each plasma cell marker, except for p18, the frequency of combined plasmacytic/plasmablastic features; the plasmacytomas positive cases and the expression levels for the positive cases of gld mutant mice, previously designated plasmacytoid lympho- increased progressively during the course of the same transitions. mas, were plasmablastic, and the IL6 transgenic cases were These results strengthened the suggestions from histologic studies plasmacytic. that plasmablastic and anaplastic plasmacytomas are reflective of Immunohistochemical analyses of GC and post-GC B- intermediate stages of normal plasma cell differentiation. lineage neoplasms. Histologic observations suggested that many Gene expression profiles of plasma cell–related neoplasms. of the nontransgenic plasmacytomas included in this study had In an earlier study, we used gene expression profiling to show features suggestive of stages in normal plasma cell differentiation that plasmacytoid lymphoma could be placed at an early stage of intermediate between immunoblasts and terminally differentiated plasma cell differentiation and that they could readily be cells. To test this idea more directly, we did immunohistochemical distinguished from other mature B-cell lineage lymphomas (21). analyses of a panel, including centroblastic diffuse large B-cell To place the plasmacytomas described here in this evolving lymphoma, immunoblastic lymphomas, plasmablastic plasmacyto- developmental scheme, we used oligonucleotide arrays that mas, and plasmacytic plasmacytomas for expression of genes that queried over 11,000 genes to characterize the expression patterns are normally expressed at high levels in B cells but are down- of immunoblastic lymphomas and the all subsets of plasma cell– regulated in mature plasma cell (PAX5, BCL6, IRF8, and PU.1) and related neoplasms (Fig. 3). The plasmacytomas of IL6 transgenic others that are normally expressed at low levels in B cells but are mice were chosen as representative of plasmacytic cases, and those up-regulated in plasma cells (IRF4, CD138, p18, XBP1, BLIMP, and labeled ‘‘APCT’’ comprised nine plasmablastic and one anaplastic immunoglobulin n light chain). Typical results obtained with a case. plasmacytic case are shown in Fig. 2. The population of mature A hierarchical clustering algorithm was used to group the tumor plasma cell seen in a section stained with H&E (Fig. 2A) showed samples, based on similarities in expression patterns of the genes, intense nuclear staining for IRF4 (Fig. 2B), membrane reactivity for and to group the genes, based on similarities in expression patterns CD138 (Fig. 2C), and cytoplasmic staining for XBP1 (Fig. 2D). across all the tumor cases. The cases segregated into two distinct Cases tested for expression of each protein were graded as groups: IL6 transgenic plasmacytic plasmacytomas in one group negative or as positive using a three-point scale to describe both and all other cases in the other. The nonplasmacytic cases

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Table 1. Immunohistochemical analyses of lymphomas and plasmacytomas for genes differentially expressed during the normal maturation of B cells from centroblasts to plasma cells

Genes Centroblastic Immunoblastic Anaplastic/plasmablastic Plasmacytic lymphoma (n = 10) lymphoma (n = 10) plasmacytoma (n = 30) plasmacytoma (n = 10)

Expression level Expression level Expression level Expression level

Positive +++ ++ + Positive +++ ++ + Positive +++ ++ + Positive +++ ++ + (%) (%) (%) (%)

PAX5 100 3 5 2 0 80 1 5 2 2 33 0 0 10 20 0 0 0 0 10 BCL-6 80 6 2 0 2 90 6 3 1 1 20 0 0 6 24 0 0 0 0 10 IRF8 80 5 2 1 2 20 0 0 2 8 17 0 0 5 25 0 0 0 0 10 PU.1 80 5 3 0 2 30 0 0 3 7 20 0 0 6 24 10 0 0 1 9 IRF4 20 0 0 2 8 50 0 2 3 5 96 5 16 8 1 100 8 2 0 0 CD138 20 0 0 2 8 20 0 0 2 8 96 1 12 16 1 100 9 1 0 0 P18 20 0 0 2 8 20 0 0 2 8 10 0 0 3 27 20 0 0 2 8 XBP1 10 0 0 1 9 10 0 0 1 9 80 1 6 17 6 100 5 5 0 0 BLIMP 0 0 0 0 10 10 0 0 1 9 80 1 2 21 6 100 0 2 8 0 KAPPA 100 0 1 9 0 100 0 3 7 0 100 10 10 10 0 100 2 7 1 0

comprised two major subsets, one of which encompassed all SJL cases being more related to the immunoblastic lymphomas and and two plasmacytoid lymphoma cases. The second subset was NFS.V+ plasmablastic cases than to the plasmacytic plasmacytomas made up of all immunoblastic lymphomas, all the plasmablastic of the IL6 trangenics. cases, and four plasmacytoid lymphoma cases. These results Of interest, the immunoblastic lymphomas and new plasma- indicted that the plasmablastic cases were most closely aligned blastic plasmacytomas segregated into two compartments of near- with immunoblastic lymphomas and some plasmacytoid lympho- equal size. A review of the histologic features of all these cases ma cases, with the remaining plasmacytoid lymphoma and the SJL revealed no defining differences between the subsets, although the

Figure 3. Relations of immunoblastic lymphomas and plasma cell–related neoplasms as determined by gene expression profiling, hierarchical clustering of histologically defined cases, and gene expression. Dendrogram at the top, the samples studied and their relationships based on similarities in gene expression. Dendrogram at the right, the expression patterns of genes across all samples with intensities depicted according to the color scale (bottom). The bars under the heat map are color coded according to the histologic classification of each tumor sample. PCT, plasmacytic plasmacytoma; PL, plasmacytoid lymphoma; SJL, SJL lymphoma; APCT, anaplastic and plasmablastic PCT; IBL, immunoblastic lymphoma.

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Downloaded from cancerres.aacrjournals.org on September 28, 2021. © 2007 American Association for Cancer Research. Cancer Research anaplastic case fell with the immunoblastic lymphomas. In addition, there were no histologic features that segregated with the two subsets of immunoblastic lymphomas. The array analyses showed that Myc transcripts were greatly increased in the plasmacytic plasmacytomas of IL6 transgenic mice but that levels expressed by the other plasma cell neoplasms did not differ significantly from the levels in normal splenic B cells. The features of Myc expression identified by the arrays were confirmed by real-time quantitative reverse transcription-PCR (Supplementary Fig. S3A). The Myc phenotype of the IL6 transgenic plasmacytic plasmacytomas was consistent with the regular but not universal occurrence of cis-activating T(12;15) translocations in the B-cell neoplasms of these mice (20) and the activation of Myc in trans in cases without translocations (36). Previous studies of plasmacytoid lymphoma showed that they did not exhibit structural alterations in the Myc locus detectable by Southern analyses and did not express Myc transcripts at high levels (18). The data from studies of Myc transcripts in immunoblastic lymphomas and SJL lymphomas and our newly described cases could thus be interpreted to suggest that Myc translocations were unlikely to be involved in the pathogenesis of those tumors. To test this prediction for the plasmablastic plasmacytomas, DNA was tested by Southern blotting for structural alterations in Myc. DNA from a plasmacytoma of a Myc knockin mouse (36) and a plasmablastic plasmacytoma of a n-MYC transgenic mouse served as positive controls (Fig. 4, bottom). The results showed that none of 12 plasmablastic nontransgenic plasmacytomas had a structural change in Myc (Fig. 4 and data not shown). The failure to detect structural changes in Myc in our neoplasms could not be ascribed to polyclonality, as each exhibited clonal rearrangements of IgH loci (Fig. 4, top; and data not shown). Studies of sections for T(12;15) by tissue FISH identified translocations in two IL6 transgenic plasmacytomas but not in any of five NFS.V+ or three SJL cases (Supplementary Fig. S3B). To gain a broader perspective on the differences that distinguish plasmablastic from plasmacytic plasmacytomas, we identified a set of genes that differ in expression by 4-fold between the two subsets. Figure 4. Organization of IgH and Myc loci by Southern blot analysis. DNA from The results of these analyses showed that the two groups of normal spleen (Normal), a plasmacytic plasmacytoma (PCT), and nine cases of plasmacytomas differed in the expression of cell surface markers plasmablastic and anaplastic plasmacytomas (1–9) were digested with defining lineage, state of differentiation, and signaling pathways appropriate enzymes and hybridized to an IgH JH probe (A)oraMyc exon 2 probe (B). (Table 2; data not shown). Among others, the plasmablastic cases preferentially expressed a variety of mature B-cell genes (Cd19, Mta3), cytokine receptors (Il5ra,), and genes involved in BCR Not surprisingly, the gene group that characterized the signaling (Cd79a, Blk), growth, survival, and differentiation plasmacytic cases included many governing protein metabolism (Tnfsf13b, Il21r). The high level of mitotic activity that characterizes and stability as well as transcriptional activity. Xbp1, which plasmablastic plasmacytomas was reflected in the elevated expres- encodes a protein required for the unfolded protein response of sion of receptor and nonreceptor tyrosine kinases, the activity of G mature plasma cells, was prominent among the transcription protein and other signaling components, and the overrepresenta- factors. It is noteworthy that a number of other genes commonly tion of transcription factors. In keeping with the high apoptotic associated with terminal B-cell differentiation-Prdm1 (BLIMP1), index of the plasmablastic cases, they expressed many more genes Sdc1 (Syndecan), and Irf4-did not appear in the plasmacytic series, involved in regulating apoptosis than did the plasmacytic cases. Of indicating that they were expressed at relatively similar levels by interest, the plasmablastic cases expressed a series of genes the two sets of plasmacytomas. suggesting active involvement of T cells (Cd3d, Cd3e, Cd4), natural killer cells (Klrd1), and macrophages as well as genes involved in inflammatory responses not seen with the plasmacytic cases. Discussion Among other elements involved in cell-matrix interactions, the It is well established that different classes of mature B-cell plasmablastic cases exhibited expression of a large number of inte- neoplasms exhibit features that mimic specific stages of normal B- grins, whereas the plasmacytic cases featured a substantial number cell differentiation and that these similarities provide a perspective of collagen genes. Finally, there were significantly more genes important to their classification and nomenclature. The studies encoding cytoskeletal proteins for plasmablastic than plasmacytic presented here define an ordered spectrum of transformed cells plasmacytomas. that mirror the stages by which normal immunoblasts gradually

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Downloaded from cancerres.aacrjournals.org on September 28, 2021. © 2007 American Association for Cancer Research. Anaplastic, Plasmablastic, and Plasmacytic Plasmacytoma assume the mantle of mature plasma cells. We have classified lymphoma cases but less mature than the plasmacytoma cell lines plasmacytomas of increasing maturity as anaplastic, plasmablastic, analyzed in one study (21), our data suggest that the fate of normal and plasmacytic to provide a parallel with the classification cells committed to the plasma cell pathway is mirrored within the systems used for human plasmacytoma and multiple myeloma and neoplastic progression of immunoblastic lymphomas ! anaplastic because of the ease with which the mouse variants could be plasmacytomas ! plasmablastic plasmacytomas ! plasmacytoid assigned to parallel subtypes. lymphoma ! SJL ! plasmacytic plasmacytomas. The results from immunophenotypic studies clearly placed This pathway will certainly deviate from that followed by normal plasmablastic plasmacytomas at a stage intermediate between B cells, because certain features of the tumors will reflect immunoblastic lymphomas and plasmacytic plasmacytomas of IL6 mechanisms involved in their transformation or interactions with transgenic mice. Analyses of gene expression profiles of these and other cell types and noncellular stromal elements. This is best other plasma cell–related malignancies, including plasmacytoid exemplified by the high levels of Myc expressed by the mostly lymphoma and SJL disease, refined this assignment by showing the plasmacytic pristane-induced plasmacytomas of BALB/c mice and plasmablastic plasmacytomas to be more closely related to the plasmacytic plasmacytomas of IL6 transgenic mice studied immunoblastic lymphomas than the other types. In light of earlier here compared with the negligible levels of Myc expressed by findings that SJL cases were more mature than plasmacytoid normal plasma cell. In almost all cases of plasmacytomas induced

Table 2. Genes that best discriminate plasmablastic from plasmacytic plasmacytomas as determined by microarray analyses

Plasmablastic Plasmacytic

B-cell signaling/immunoglobulin Blk, Brdg1, Card11, Cd19, Cd22, Cd24, Cd40, Cd79a, Dapp1, Blnk Dntt, Fcer2, Ly86, Mta3, Nfam1, Raftlin, Rag1, Rgs1, Vav1 Cytokines/growth factors Cish, Igf1, Il1r2, Il2rb, Il2rg, Il4r, Il5ra, Il12a, Il17rb, Il18, Il18bp, Bmp1, Igfbp3, Inhbb, Il18rap, Il20, Il21r, Il27ra, Pdgfb, Plac8, Socs1, Tgfb1, Tnf, Jub, Pdgfra, Vegf Tnfaip2, Tnfaip3, Tnfrsf14, Tnfsf11, Tnfsf13b Chemokines Ccl3, Ccl4, Ccl5, Ccl19, Ccr1, Ccr2, Ccr5, Cxcl4, Cxcl7, Xcl1 Ccl11 Receptor tyrosine kinase Axl, Epor, Flt3, Kit Nonreceptor tyrosine kinase Fgr, Hck, Limk1, Stk10, Stk39 Csnk1e, Matk, G proteins Arf5, Arhgdib, Arrb1, Centd1, Centd2, Frmd4b, Gnb4, Gng11, Gpsm3, Arhgap8, Farp2, Maged1, Mageh1, Rabep2, Rac2, Rassf2, Rassf4, Rgl1, Rgs16, Rrad, Rras2, Sh3bp1 Nol8, Pscdbp, Rabggta, Rgsl1 Signaling Adcy7, Btg1, Cnp, Dok1, Dusp2, Ehd1, Gna15, Grb2, Hcls1, Dusp22, Ick, Pik3gc, Ppp2r5a, Skil, Mobk1b, Pak1ip1, Pdlim7, Plcl2, Plek, Plxnc1, Ppbp, Pstpip1, Snd1, Stat2, Tbrg4, Rob1, Trib1 Ptpn1, Ptpn6, Ptpn18, Rbp4, S100a13, Sh2d1a, Sh3bp2, Sla, Slamf1, Sts-1, Tlr1, Tlr9, Traf1 Transcription factors/cofactors Abi3, Ankrd1, Batf, Bhlhb2, C2ta, Dek, Gata1, Id2, Lrrfip1, Mtpn, Aebp1, Basp1, Etv4, Figla, Myb, Mycn, Nfkbia, Nfkbie, Nr1h3, Nrob1, Rb1, Relb, Sox11, Gcn5l2, Ixl, Lztr1, Mybbp1a, Sp100, Spib, Srf, Tcfeb, Wtap, Znf318, Zfp36l1, Zfpm1, Znf238 Myc, Rnf25, Usf1, Xbp1 Apoptosis Anxa11, Apaf1, Arl6ip, Arl6ip5, Bag2, Bcl2, Casp1, Casp3, Casp4, Nol3, Park7 Casp6, Casp7, Clu, Dnase1l2, Dnase1l3, Fas, Hrk, Phlda2, Plscr1, Ptpn13, Rps6ka1, Stk17b, Tegt T cells Cd3d, Cd3e, Cd3g, Cd4, Cd7, Cd37, Cd53, Cd82, Cdc42se1, Dpp4, Icoslg, Lag3, Lat, Lck, Trat1, Tgtp, Trim30, Zap70 NK cells Cd244, Klrd1 Ctsw, Klra17, Pvr Macrophages Cd5l, Cnr2, Csf1r, Marco, Myo10, Nramp2 Inflammation/IFNs Abp1, Alox5ap, Bdkrb2, Clec4e, Ddt, Gbp1, Gbp2, Irgm, Ifi35, Ifngr1, Lbp, Ptgis Iigp1, Irf1, Irf2, Irf8, Ltb, Ptges, Ptgs1, Rnase1, Rsad2, Slc43a3 Extracellular matrix/adhesion Cd47, Cib1, Fgl2, Itga6, Itgb1, Itgb2, Itgb3, Itgb7, Lgals9, Mfap1, Agr2, Alpl, C1qbp, Chst1, Col1a1, Mmp10, Ntn1, Sema4a, Tjp3 Col4a2, Col5a2, Col6a2, Extl2, Fstl1, Itgb5, Lamb1, Lamc2, Lypd3, Mmp2, Krtha2, Loxl4, Muxc1, Pvrl4, Sparcl1, Spon1, St14, Tacstd1, Timp1, Xlkd1 Cytoskeleton Actg2, Actr2, Arpc4, Avil, Cap1, Capg, Cdc42ep3, Cnn2, Fscn1, Dnch2, Mfap2, Myh14, Rsn, Lasp1, Lcp1, Lsp1, Lst1, Marcks, Plekho1, Pdlim7, Pfn1, Plec1, Svil, Trip6, Wasl Rp2, Scin, Swap70, Tln1, Tmod3, Tmsb4x, Tmsb10, Tuba8, Tubb3, Vasp, Wdr1, Zyx Protein metabolism/ Dnajb1, Gng10, Hps3, Pcsk1n, Pcsk6, Snx2, Snx10, Tgm2 Calu, Ddost, Dnajb11, Eef1a2, Eif4a1, stability/HSP Ganab, Gne, Ipo4, Itm1, P4ha2, P4hb, Pigk, Prmt7, Sec61a1, Sec63, Senp2, Srp9, Ssb1 Ubiquitin/proteases/ Capn1, Cst7, Fbxw8, Mkrn1, Prtn3, Psmb8, Psmb9, Serpinb1, Arfip2, Bsg, Enpep, Epas1, Klk2, Os-9, protease inhibitors Trim21, Ube2d1, Ube2d2 Psmb3, Ube2t, Serpina3, Slpi, Uchl5

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Downloaded from cancerres.aacrjournals.org on September 28, 2021. © 2007 American Association for Cancer Research. Cancer Research by pristane or occurring spontaneously in transgenic mice, elevated expanded population of peritoneal B1 cells that could be the target expression of Myc is due to chromosomal translocations that for transformation rather than MZ B cells; NZB plasmacytomas juxtapose the Myc locus to IgH, T(12;15), or, less commonly, one of also have translocations involving the Myc locus. the IgL chain loci (16, 20). High-level expression of Myc in the Turning to cases of plasmacytomas without Myc translocations, absence of translocations is uncommon (16, 36). In contrast, the plasmacytoid lymphoma of autoimmune BALB/c-Fasl/Fasl structural alterations in Myc detectable by Southern analysis mutant mice exhibited an even higher ratio of IgG-producing were not detected in the anaplastic, plasmablastic, and SJL tumors to IgA-producing tumors than pristane-induced plasmacy- plasmacytomas studied here and are not a feature of plasmacytoid tomas of NZB; however, the antigenic specificities were often lymphoma (18). Furthermore, none of the plasmacytoid lymphoma, polyreactive, including anti-self (21). SJL mice are well known for SJL, and plasmablastic plasmacytoma cases studied here by their sensitivity to experimentally induced autoimmune disorders quantitative PCR exhibited elevated levels of Myc transcripts or such as experimental autoimmune encephalomyelitis, potentially exhibited T(12;15) by tissue FISH, indicating that these tumors tying them through autoimmunity to BALB/c Fasl mutant mice. did not have Myc-activating translocations involving the Pvt1 Perhaps more important, SJL mice exhibit increasing splenomegaly region (16) that would have escaped detection by our Southern and lymphadenopathy with age, due in large part to the expansion analyses. of lymphoid follicles with large, active GC. This phenotype is also The Myc-independent mechanisms involved in transformation of true of NZB and Fasl/Fasl mutant mice. In SJL and NZB mice, the plasma cell–related cases described here and previously (36) plasma cells can sometimes be seen to spill from the enlarged, are not known but are of considerable interest. Given the different active GC and may be the precursors to plasmacytoma. If the ‘‘non- routes that can give rise to normal plasma cell maturation of B1 Myc phenotype’’ of the plasmacytoid lymphoma, SJL, and NFS.V+ cells, extrafollicular foci, and products of GC reactions, it may be plasmacytomas reflects a GC origin, the declension of Myc-driven that Myc-dependent and Myc-independent plasmacytoma subsets versus non–Myc-driven plasmacytoma may provide direction for have different cellular origins. B1 cells might be the cell of origin of further experimental studies. A pristane-induced plasmacytomas and those of iMycE and IL6 The range of cytologic variation for mouse plasmacytoma transgenic mice. This notion stems from several facts. First, the described here was first recognized in a review of cases occurring vast majority of pristane-induced cases express IgA (16, 37). in EA-v-abl transgenic mice (19, 44) but has also been seen in A Second, the plasmacytomas of iMycE and IL6 transgenic, which studies of a number of other strains of genetically engineered and are mostly IgG producing, develop preferentially in gut-associated conventional inbred mice, including the published studies of IL6 A lymphoid tissues (20), whereas pristane-induced tumors arise from transgenic (20) and iMycE knockin mice (22, 23). This strongly peritoneal granulomas (16). Third, pristane-induced plasmacyto- suggests that the conclusions we have drawn can be generalized to mas are similar to B1 cells in expressing antibodies to a variety of other settings in mice and may be relevant for understanding polysaccharide or other repeating antigens as well as autoreactive human plasma cell neoplasms. polyspecific antibodies that are similar to ‘‘natural’’ antibodies of It is important to note that our observations were made only at the normal immune repertoire (38). Finally, BALB/c mice that carry necropsy. This leaves open the question of whether the plasma- the xid mutation in Btk, and as a consequence are devoid of B1 blastic and anaplastic plasmacytomas arose de novo or represent a cells, are strikingly resistant to pristane-induced plasmacytoma progression from a low-grade, more differentiated plasmacytic induction (39). malignancy. Studies of patients with multiple myeloma showed Extrafollicular foci of plasma cell can develop from antigen- that some progressed from a plasmacytic type to a clonally related, stimulated follicular B cells (40) or marginal zone B cells (41). The more aggressive form characterized by the presence of proliferating marginal zone B-cell subset has many features in common with B1 immunoblasts of varying sizes with amphophilic cytoplasm and cells that include overlapping repertoires and favored isotypes, thickened nuclear membranes (45, 46), features seen with regularity similarly heightened sensitivity to proliferative stimuli, and in our anaplastic and plasmablastic subsets of plasmacytoma. prominent contributions to T-independent responses (42). In Progression from a low-grade lymphoma to immunoblastic addition, the marginal zones of mice bearing the xid mutation lymphoma is also characteristic of the emergence of Richter’s are scantly populated by B cells (43). The many parallels between syndrome in patients with chronic lymphocytic leukemia (47), B1 and marginal zone B cells and their plasma cell progeny make it which seems to be a malignancy of memory B cells. Although difficult to favor plasma cell derived from one subset versus these results could be interpreted as the manifestations of de- the other as the normal forerunners of pristane-induced, IL6 differentiative processes, studies of cells from patients with A transgenic, or iMycE plasmacytomas. If this model is correct, it multiple myeloma have suggested otherwise. In particular, the remains to be determined why overexpression of Myc would result data of Matsui et al. (48) indicate that the cell type responsible for in the seemingly selective transformation of these cells. the initiation and maintenance of multiple myeloma is a minor Of interest, previous studies of autoimmune NZB mice, the only population of proliferative post-GC CD19+CD20+sIg+CD138 B cells strain besides BALB/c to be highly susceptible to plasmacytoma with the capacity to differentiate into the mass of CD138+ plasma induction by pristane (17) and to exhibit much enlarged marginal cells that comprise the bulkof the disease. This phenotype is zones, suggested that the cell population susceptible to transfor- similar to that of cells comprising anaplastic and plasmablastic mation by pristane was different from that in BALB/c. This view plasmacytomas, respectively. was based on the finding that the frequency of IgG-producing Further analyses of mouse plasmacytomas of different origins plasmacytomas was more than twice as high and that the may enhance our understanding of the relations between mouse frequency of plasmacytomas producing IgA was less that half that and human plasma cell–related tumors, help define the transitions observed with BALB/c. In addition, few NZB plasmacytomas involved in the later stages of normal B-cell differentiation, and secreted antibody specific for the antigens bound most frequently provide experimental models for understanding the nature of stem by BALB/c plasmacytomas; however, NZB mice also have an cells that are postulated to drive these diseases.

CancerRes 2007; 67: (6). March15, 2007 2446 www.aacrjournals.org

Acknowledgments This article is dedicated to the memory of the late Alan W. Harris. We thank Dr. Alfonso Macias for many contributions to the study, the NIAID intramural Received 5/2/2006; revised 8/22/2006; accepted 12/27/2006. editor Brenda Rae Marshall for excellent editorial assistance, Nicole McNeal Grant support: Intramural Research Program of the NIH, NIAID, and NCI grants (Genetics Branch, Center for Cancer Research, NCI) and Dr. Makiko Takizawa DK56597 and CA34196 (D.C. Roopenian) and CA82872-02 (W.F. Davidson). (Molecular Immunology and Inflammation Branch, NIAID) for assistance with The costs of publication of this article were defrayed in part by the payment of page the FISH technique, and Dr. Sabine Mai (Genomic Center for Cancer Research charges. This article must therefore be hereby marked advertisement in accordance and Diagnosis, Manitoba Institute for Cell Biology) for helpful technical tips with 18 U.S.C. Section 1734 solely to indicate this fact. on FISH.

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