Gene Expression Analysis of Plasmablastic Lymphoma Identifies

Letters to the Editor 2270 Gene expression analysis of plasmablastic lymphoma identiﬁes downregulation of B-cell receptor signaling and additional unique transcriptional programs

Leukemia (2015) 29, 2270–2273; doi:10.1038/leu.2015.109 two of which (PBL_13 and PBL_15) clustered with EOP and the other two (PBL_6 and PBL_8) clustered with DLBCL. The latter two PBL cases exhibited characteristic PBL morphology with the Plasmablastic lymphoma (PBL) is a distinct aggressive B-cell following immunophenotypes: CD79a+CD138- MUM1+PAX5- malignancy characterized by a terminally differentiated B-cell CD20-EBER-(PBL_6) and CD79a+CD138+MUM1+PAX5-CD20- EBER+ phenotype. It usually occurs in immunocompromised patients, CD10+ (PBL_8) and lacked expression of pan B-cell markers CD20 either in the setting of HIV infection or in patients with iatrogenic and PAX5. Overall, gene expression profiles of PBLs were more immunosuppression and advanced age.1,2 Histopathologic features similar to the EOPs than DLBCLs, likely owing to the origin of both of PBL include immunoblastic and/or plasmablastic morphology, PBLs and EOPs from plasma cells. high proliferation rate and immunophenotypic evidence of terminal We focused our analysis on the differences between PBL and B-cell differentiation.1,3 Other features include decreased expression DLBCL, and identified 645 genes as differentially expressed between of B-cell lineage antigens4 and frequent infection with Epstein– them (FDRo1%; Figure 2a, Supplementary Methods and Barr virus (EBV).5 The molecular and genetic mechanisms under- Supplementary Table 2), with 257 highly expressed in PBL relative lying its pathogenesis remain largely unknown. Although EBV to DLBCL, and 388 lower in PBL. Gene expression data were infection may contribute, it does not account for lymphomagenesis selectively validated by real-time PCR for three candidate genes in EBV-negative cases. Among the most frequently detected (LYN, SPIB,andSWAP70), chosen on the basis of differential fold genomic changes are chromosomal translocations involving the change between DLBCLs and PBLs. RT–PCR results were highly MYC gene, usually in association with the immunoglobulin heavy correlated with the array data (R2 = 0.523–0.737; not shown). chain, detected in 49% of cases.6 However, whether MYC Transcriptional factors BCL6, BCL11A and SPI-B, implicated in the – translocations represent initiating or late genetic events in PBL pathogenesis of DLBCLs,8 11 showed significantly (FDRo0.01 in pathogenesis is unknown. The limited knowledge of PBL pathogen- all cases) lower mRNA expression in PBLs. A similar trend was esis may contribute to the absence of effective targeted therapeutic observed for CXCR5 and IL4, which have a role in the germinal approaches, and thus to the poor outcome of PBL patients, who center (GC) microenvironment. These changes likely reflect post- have an estimated 2-year overall survival below 40%.1,7 GC-plasmablastic origin of these tumors. In contrast, mRNA levels We profiled gene expression in PBL cases from nine male and of PRMT5, an important transcriptional repressor, were signifi- six female patients with a median age of 40 years (range 4–60 cantly higher in PBLs than in DLBCLs (FDRo0.01, twofold years). HIV infection was present in 60% of the patients, and difference on average). IL21 induces direct cytotoxicity in DLBCLs tumors predominantly originated from the extranodal sites but not in neoplastic plasma cells.12 Interestingly, IL21R mRNA (Supplementary Table 1). Five of the 15 cases comprised sheets expression was similar in PBLs and DLBCLs. of large immunoblastic and/or plasmablastic cells with only We identified biological pathways and themes underlying the occasional plasmacytic features (plasmablastic PBL, Figure 1a). malignancy-specific gene expression patterns by comparison with Ten tumors were composed predominantly of sheets of immuno- previously defined gene sets (Supplementary Table 3).13 Genes blasts or plasmablasts with easily discernible plasmacytic differ- highly expressed in primary effusion lymphomas (PELs) were also entiation (plasmacytic PBL, Figure 1b). Evidence of plasma cell high in PBLs vs DLBCLs (see below). Many B-cell receptor signaling differentiation was identified in all cases by immunohistochem- pathway genes were significantly (FDRo0.001, hypergeometric istry (IHC) (Supplementary Table 1). EBV was detected by in situ test) expressed at lower levels in PBLs relative to DLBCLs. hybridization in 60% of cases (Figure 1c) and strongly correlated Individual BCR signaling genes (CD79A, CD79B, BLK, LYN, SYK, with HIV infection. FISH detected MYC translocations in 2 of 4 PTPRC, CSK, PIK3CD, SWAP70, and REL) were repressed by twofold analyzed cases, and MYC protein expression was detected by IHC or more on average in PBLs (Figure 2b, Supplementary Table 3). In in 12/15 cases. We also profiled 10 DLBCL cases negative for EBER contrast, mitochondrial genes (ATP5G1, ACADVL, CLPP, CYC1, by in situ hybridization and five extramedullary (extraosseous) FARSA, FH, LONP1, LYPLA2, MRPS2, MRPL12, MRPL40, NDUFAF1, plasmacytoma (EOP). These tumors were chosen for comparison NDUFB6, NDUFB7 SEC61G, SLC25A28 TUFM and UQCRQ; with PBL, as they represent a spectrum of neoplasms originating Supplementary Table 3) were higher in PBLs. Targets of MYB, a from cells representing sequential stages in B-cell differentiation. major transcriptional regulator of hematopoietic differentiation,14 The DLBCL tumors were defined as germinal center B-cell (GCB)- were upregulated in PBLs (Figure 2a and Supplementary Table 3). like (five tumors) and activated B-cell-like (five tumors) DLBCLs by Transcriptionally activated targets of NF-KB signaling in multiple expression profiling. MYC protein expression was detected by IHC cell types (HeLa cells,15 fibroblasts,16 keratinocytes,16 and mela- in six of nine tested DLBCLs. EOPs included in this study were all noma cell lines17) were expressed at lower levels in PBLs isolated, extraosseous (extramedullary), plasma cell neoplasms, (Supplementary Table 3), which was confirmed by analysis of a and were well-differentiated neoplasms composed of small and large compendium of sets of transcription factor targets mature plasma cells. All EOPs expressed markers of plasma cell (Supplementary Table 4). MYC RNA was expressed in all PBL differentiation (for example, CD138) and demonstrated cytoplas- samples, and MYC protein was expressed in 12 of 15 analyzed PBL mic light-chain restriction, as determined by IHC for kappa and tumors. The percentage of MYC protein-positive cells was highly lambda light chains. correlated with MYC RNA expression (ρ = 0.76, P = 0.004; When clustered hierarchically (Supplementary Methods), sam- Supplementary Figure 1). Genes upregulated upon MYC ples largely grouped according to histopathologic type, not by knockdown in Burkitt lymphoma18 were significantly enriched primary organ of origin (Figure 1d). The exceptions were four PBLs, among those higher in DLBCLs vs PBLs (P =2×10− 39,

Accepted article preview online 24 April 2015; advance online publication, 19 May 2015

50 µM 50 µM 100 µM 0 1 2 -2 -1 PBL_1 PBL_2 PBL_3 PBL_4 PBL_7 PBL_10 PBL_11 PBL_9 PBL_5 PBL_12 PBL_14 PBL_13 PBL_15 EOP_26 EOP_27 EOP_28 EOP_29 EOP_30 PBL_6 PBL_8 DLBCL_16 DLBCL_20 DLBCL_23 DLBCL_17 DLBCL_19 DLBCL_22 DLBCL_21 DLBCL_18 DLBCL_24 DLBCL_25 Figure 1. Histologic and gene expression features of PBLs. (a) Immunoblastic and/or plasmablastic morphology were observed in all PBL cases, hematoxylin and eosin (H&E) staining, × 400 magnification. (b) Cases of PBL were designated as having plasmacytic morphology when they were composed predominantly of sheets of large plasmacytoid cells with a more mature morphologic appearance, with a minority of cells having an immunoblastic and plasmablastic appearance, H&E staining, × 400 magnification. (c) EBV positivity by in situ hybridization in a plasmacytic PBL, EBER staining by in situ hybridization, × 200 magnification. (d) A total of 3190 genes with significant variation across samples were clustered hierarchically. With the exception of two PBL samples (PBL_6 and PBL_8) that clustered with DLBCLs, and two that clustered with EOPs (PBL_13 and PBL_15), the different malignancies grouped separately. hypergeometric test; SEITZ_MYC_ko_UP in Supplementary Table PELs and DLBCLs21 (Supplementary Methods). One thousand two 4; Supplementary Figure 2). Consistently, genes downregulated by hundred and fifty-two genes were differentially expressed in PELs vs MYC knockdown were high in PBLs (SEITZ_MYC_ko_DOWN, PBLs (FDR 0.5%; Supplementary Figure 3, Supplementary Table 5). P =3×10− 18; Supplementary Figure 2). These results remained The top 50 genes most overexpressed in PELs relative to PBLs true after filtering for genes also bound by MYC according included 11 (CFI, GZMA,PPAP2B, IL10, IL2RB, MUC1, IQGAP2, LTA4H, to ChIP-Seq (SEITZ_MYC_ko_UP_and_CHIP, P =2×10− 22, and DAZL, TNFRSF8, ALDH1L1)previouslyidentified as high in PELs SEITZ_MYC_ko_DOWN_and_CHIP, P =6×10− 17; Supplementary compared with other B-cell lymphomas.22 Table 4 and Supplementary Figure 2).19 Genes repressed by We compared our PBLs and DLBCLs with a larger series of 200 MYC in a MYC-driven transgenic mouse model of B-cell lymphoma DLBCLs8 (Supplementary Figure 4). All except one of our cases of were also among those low in PBLs relative to DLBCLs20 PBLs clustered together and separately from the DLBCLs. (YU_MYC_TARGETS_DN, Supplementary Table 3). However, PBLs were more similar to activated B-cell DLBCLs than Eight of the EBV+ cases were HIV+, whereas all EBV − cases GCB DLBCLs, as might be predicted from the corresponding were HIV − (Supplementary Table 1). No individual genes cell of origin. Genes high in plasma cells relative to other types of emerged as significant (FDR 10%) based on two-class SAM B cells were high in PBLs compared with DLBCLs (Supplementary analysis of EBV+ vs EBV − PBL, indicating that EBV and HIV Figure 5),23 as were the transcription factor XBP1, a critical infection do not significantly affect transcriptional patterns regulator of plasma cell differentiation24 and the plasma cell in PBLs. marker CD138 (Figure 2a). We observed low expression in PBLs of We also compared our PBLs with gene expression profiles of PELs other transcriptional factors regulating the mature B-cell tran- – another tumor originating from plasmablasts and commonly scriptome (BCL6, PAX5, SPI-B) and upregulation of BLIMP1, XBP1 infected with EBV – by merging our data with a series containing and IL21R, which regulate differentiation to plasma cells.11,24

PBL DLBCL PBL DLBCL MS4A1 SPIB FC

2 CD22 BCL6 CD19 SWAP70

Mean log BCL6

BCL11A CD72 SWAP70 CXCR5 BCL2A1 LCK BLK LYN PIK3CD REL NFKB1 targets PTPRC

BCR signaling pathway 2 CD79A 1 0 ETS1 -1 -2 SYK RASGRP1 IL7 PRMT5 BLNK IFNG CCNB1 CD81 CD320 RASGRP3 CD300A PRKCB

High in plasma cells, IL6R FN1 MYC targets, MYB targets DNMT3B BCL2 CD138 OSM IL2

100 µM 100 µM 100 µM Figure 2. Differentially expressed genes in PBLs. (a) A total of 645 genes were significantly differentially expressed (FDRo1%) between PBLs and DLBCLs. Approximate positions of individual genes discussed in the text are indicated. The first column shows the average log2 fold- change between PBLs and DLBCLs. Significant overlaps of clusters with MYC, MYB and NFKB1 targets are shown, as well as the BCR signaling pathway and genes that are more highly expressed in plasma cells than in other B lymphocytes. (b) Differentially expressed genes between PBLs and DLBCLs included the ones encoding members of the BCR signaling pathway that were generally more highly expressed in DLBCLs. (c) Positive staining of SWAP70 in GC B cells in a normal tonsil and negative staining of surrounding marginal zone B cells and paracortical T cells served as positive and negative controls, respectively. (d) Representative example of immunopositivity for SWAP70 in DLBCL. (e) Representative example of immunonegativity for SWAP70 in PBLs.

These observations are in concordance with IHC data in our, and 45 DLBCLs, all PBLs were CD138+CD20-EBER+, whereas DLBCLs previously reported, PBL cases.2,4 (24 GCB type, 21 non-GCB) were uniformly CD20+CD138 − . Three To validate differences in gene expression at the protein level of these DLBCLs (all non-GCB subtype) were EBER-positive. Only 1 and to identify potential IHC markers that could be used for of the 12 additional cases of PBLs was positive for SWAP70 protein differential diagnosis in routine practice, we focused on the BCR expression, whereas all 45 DLBCLs were positive. signature gene SWAP70, which was downregulated in PBLs in The majority of previously reported aberrations are not unique comparison with DLBCLs, and could contribute to differential to PBLs. We found that PBL has a transcriptional profile distinct diagnosis in routine practice. In normal B-cell differentiation, from DLBCL with differences in activation of BCR signaling SWAP70 mRNA is expressed most highly in GC B cells and is low in (possibly reflecting their origin from plasmablasts) and targets of plasma cells (Supplementary Figure 6). IHC for SWAP70 protein the transcription factors MYC and MYB. These features support confirmed its expression in GC B cells in normal tonsils (Figure 2c). classification of PBL as a distinct entity and warrant further studies SWAP70 was not detected by IHC in all 25 PBL cases analyzed by examining the role for MYC and MYB in the pathogenesis of PBL. arrays, but it was expressed in all 10 DLBCLs. All DLBCLs We also identified potential biomarkers (for example, SWAP70) demonstrated diffuse, strong cell membrane and cytoplasmic that could be useful for pathological differentiation of aggressive positivity in greater than 90% of tumor cells (Figures 2d and e). In lymphomas by IHC. Chopin et al. have shown that SWAP70 affects a separate validation set of 12 PBLs (Supplementary Table 6) and the expression of IRF-4, which is in turn required for the

Leukemia (2015) 2257 – 2273 © 2015 Macmillan Publishers Limited Letters to the Editor 2273 differentiation of mature B cell into an antibody-secreting plasma 6 Valera A, Balague O, Colomo L, Martinez A, Delabie J, Taddesse-Heath L et al. cell.25 Interestingly, SWAP70s effects on differentiation to plasma IG/MYC rearrangements are the main cytogenetic alteration in plasmablastic cells appear to occur early in the plasma cell commitment process. lymphomas. Am J Surg Pathol 2010; 34: 1686–1694. The significant downregulation of SWAP70 in PBLs, relative to 7 Castillo JJ, Furman M, Beltran BE, Bibas M, Bower M, Chen W et al. Human immunodeficiency virus-associated plasmablastic lymphoma: poor DLBCLs, suggests that these cells are already well committed to 118 plasma cell differentiation. prognosis in the era of highly active antiretroviral therapy. Cancer 2012; : 5270–5277. 8 Lenz G, Wright GW, Emre NC, Kohlhammer H, Dave SS, Davis RE et al. Molecular CONFLICT OF INTEREST subtypes of diffuse large B-cell lymphoma arise by distinct genetic pathways. Proc Natl Acad Sci USA 2008; 105: 13520–13525. The authors declare no conflict of interest. 9 Saito M, Gao J, Basso K, Kitagawa Y, Smith PM, Bhagat G et al. A signaling pathway mediating downregulation of BCL6 in germinal center B cells is blocked by BCL6 gene alterations in B cell lymphoma. Cancer Cell 2007; 12:280–292. ACKNOWLEDGEMENTS 10 Satterwhite E, Sonoki T, Willis TG, Harder L, Nowak R, Arriola EL et al. The BCL11 ISL is supported by Sylvester Comprehensive Cancer Center and the Dwoskin Family, gene family: involvement of BCL11A in lymphoid malignancies. Blood 2001; 98: – Recio Family and Anthony Rizzo Family Foundations. AJG is supported by 3413 3420. U54CA149145 (NCI Integrative Cancer Biology Program). FV is supported by funds 11 Shaffer AL, Lin KI, Kuo TC, Yu X, Hurt EM, Rosenwald A et al. Blimp-1 orchestrates from the Translational Grant of the Leukemia & Lymphoma Society, National plasma cell differentiation by extinguishing the mature B cell gene expression Institutes of Health K08 Physician-Scientist Award K08 CA143151-01 and the 2015 program. Immunity 2002; 17:51–62. CTSI Miami Pilot Award Program. 12 Sarosiek KA, Malumbres R, Nechushtan H, Gentles AJ, Avisar E, Lossos IS. Novel IL-21 signaling pathway up-regulates c-Myc and induces apoptosis of diffuse large B-cell lymphomas. Blood 2010; 115: 570–580. J Chapman1,8, AJ Gentles2,8, V Sujoy1, F Vega1, CI Dumur3, 3 4 5 6 13 Liberzon A, Subramanian A, Pinchback R, Thorvaldsdottir H, Tamayo P, TL Blevins , L Bernal-Mizrachi , M Mosunjac , A Pimentel , Mesirov JP. Molecular signatures database (MSigDB) 3.0. Bioinformatics 2011; 27: 6 6,7 D Zhu and IS Lossos 1739–1740. 1 Division of Hematopathology, Department of Pathology, University 14 Greig KT, Carotta S, Nutt SL. Critical roles for c-Myb in hematopoietic of Miami, Miami, FL, USA; progenitor cells. Semin Immunol 2008; 20: 247–256. 2Stanford Center for Cancer Systems Biology, Stanford University, 15 Tian B, Nowak DE, Jamaluddin M, Wang S, Brasier AR. Identification of direct Stanford, CA, USA; genomic targets downstream of the nuclear factor-kappaB transcription 3 factor mediating tumor necrosis factor signaling. J Biol Chem 2005; 280: Virginia Commonwealth University, Richmond, VA, USA; – 4Derpartment of Hematology and Medical Oncology, Winship Cancer 17435 17448. 16 Hinata K, Gervin AM, Jennifer Zhang Y, Khavari PA. Divergent gene regulation and Institute, Emory University, Atlanta, GA, USA; 5 growth effects by NF-kappa B in epithelial and mesenchymal cells of human skin. Department of Pathology, Emory School of Medicine, Oncogene 2003; 22:1955–1964. Atlanta, GA, USA; 17 Schon M, Wienrich BG, Kneitz S, Sennefelder H, Amschler K, Vohringer V et al. 6 Division of Hematology-Oncology, Department of Medicine, KINK-1, a novel small-molecule inhibitor of IKKbeta, and the susceptibility of University of Miami and Sylvester Comprehensive Cancer Center, melanoma cells to antitumoral treatment. J Natl Cancer Inst 2008; 100:862–875. Miami, FL, USA and 18 Seitz V, Butzhammer P, Hirsch B, Hecht J, Gutgemann I, Ehlers A et al. Deep 7Department of Molecular and Cellular Pharmacology, University of sequencing of MYC DNA-binding sites in Burkitt lymphoma. PloS One 2011; 6: Miami, Miami, FL, USA e26837. E-mail: [email protected] 19 Zhou L, Picard D, Ra YS, Li M, Northcott PA, Hu Y et al. Silencing of 8 thrombospondin-1 is critical for myc-induced metastatic phenotypes in medul- These authors contributed equally to this work. loblastoma. Cancer Res 2010; 70: 8199–8210. 20 Yu D, Cozma D, Park A, Thomas-Tikhonenko A. Functional validation of genes implicated in lymphomagenesis: an in vivo selection assay using a Myc-induced REFERENCES B-cell tumor. Ann N Y Acad Sc 2005; 1059:145–159. 1 Castillo J, Pantanowitz L, Dezube BJ. HIV-associated plasmablastic lymphoma: 21 Basso K, Margolin AA, Stolovitzky G, Klein U, Dalla-Favera R, Califano A. Reverse lessons learned from 112 published cases. Am J Hematol 2008; 83: 804–809. engineering of regulatory networks in human B cells. Nat Genet 2005; 37: 2 Delecluse HJ, Anagnostopoulos I, Dallenbach F, Hummel M, Marafioti T, Schneider 382–390. U et al. Plasmablastic lymphomas of the oral cavity: a new entity associated with 22 Klein U, Gloghini A, Gaidano G, Chadburn A, Cesarman E, Dalla-Favera R et al. the human immunodeficiency virus infection. Blood 1997; 89: 1413–1420. Gene expression profile analysis of AIDS-related primary effusion lymphoma (PEL) 3 Maestre L, Tooze R, Canamero M, Montes-Moreno S, Ramos R, Doody G et al. suggests a plasmablastic derivation and identifies PEL-specific transcripts. Blood Expression pattern of XBP1(S) in human B-cell lymphomas. Haematologica 2009; 2003; 101: 4115–4121. 94: 419–422. 23 Tarte K, Zhan F, De Vos J, Klein B, Shaughnessy J. Gene expression profiling of 4 Montes-Moreno S, Gonzalez-Medina AR, Rodriguez-Pinilla SM, Maestre L, plasma cells and plasmablasts: toward a better understanding of the late stages Sanchez-Verde L, Roncador G et al. Aggressive large B-cell lymphoma with of B-cell differentiation. Blood 2003; 102:592–600. plasma cell differentiation: immunohistochemical characterization of plasmablastic 24 Reimold AM, Iwakoshi NN, Manis J, Vallabhajosyula P, Szomolanyi-Tsuda E, lymphoma and diffuse large B-cell lymphoma with partial plasmablastic phenotype. Gravallese EM et al. Plasma cell differentiation requires the transcription factor Haematologica 2010; 95:1342–1349. XBP-1. Nature 2001; 412:300–307. 5 Kaplan LD. Human herpesvirus-8: Kaposi sarcoma, multicentric Castleman disease, 25 Chopin M, Chacon-Martinez CA, Jessberger R. Fine tuning of IRF-4 expression by and primary effusion lymphoma. Hematology Am Soc Hematol Educ Program 2013; SWAP-70 controls the initiation of plasma cell development. Eur J Immunol 2011; 2013: 103–108. 41: 3063–3074.

Supplementary Information accompanies this paper on the Leukemia website (http://www.nature.com/leu)