A super-SILAC based proteomics analysis of diffuse large B-cell lymphoma-NOS patient samples to identify new proteins that discriminate GCB and non-GCB lymphomas

Autoři: L. E. van der Meeren aff001;  J. Kluiver aff001;  B. Rutgers aff001;  Y. Alsagoor aff001;  P. M. Kluin aff001;  A. van den Berg aff001;  L. Visser aff001
Působiště autorů: Department of Pathology and Medical Biology, University of Groningen, University Medical Centre Groningen, Groningen, The Netherlands aff001;  Department of Pathology, University Medical Centre Utrecht, Utrecht, The Netherlands aff002
Vyšlo v časopise: PLoS ONE 14(10)
Kategorie: Research Article
doi: https://doi.org/10.1371/journal.pone.0223260


Diffuse large B-cell lymphoma—not otherwise specified (DLBCL-NOS) is a large and heterogeneous subgroup of non-Hodgkin lymphoma. DLBCL can be subdivided into germinal centre B-cell like (GCB) and activated B-cell like (ABC or non-GCB) using a gene-expression based or an immunohistochemical approach. In this study we aimed to identify additional proteins that are differentially expressed between GCB and non-GCB DLBCL. A reference super-SILAC mix, including proteins of eight B-cell lymphoma cell lines, was mixed with proteins isolated from seven non-GCB DLBCL and five GCB DLBCL patient tissue samples to quantify protein levels. Protein identification and quantification was performed by LC-MS. We identified a total of 4289 proteins, with a four-fold significant difference in expression between non-GCB and GCB DLBCL for 37 proteins. Four proteins were selected for validation in the same cases and replication in an independent cohort of 47 DLBCL patients by immunohistochemistry. In the validation cohort, we observed a non-significant trend towards the same differential expression pattern as observed in the proteomics. The replication study showed significant and consistent differences for two of the proteins: expression of glomulin (GLMN) was higher in GCB DLBCL, while expression of ribosomal protein L23 (RPL23) was higher in non-GCB DLBCL. These proteins are functionally linked to important pathways involving MYC, p53 and angiogenesis. In summary, we showed increased expression of RPL23 and decreased expression of GLMN in non-GCB compared to GCB DLBCL on purified primary DLBCL patient samples and replicated these results in an independent patient cohort.

Klíčová slova:

Algorithms – B cells – Cell staining – Immunohistochemical analysis – Immunohistochemistry techniques – Protein expression – Proteomic databases – Proteomics


1. Lenz G, Wright GW, Emre NCT, Kohlhammer H, Dave SS, Davis RE, et al. Molecular subtypes of diffuse large B-cell lymphoma arise by distinct genetic pathways. Proc Natl Acad Sci USA. 2008 Sep 9;105(36):13520–5. doi: 10.1073/pnas.0804295105 18765795

2. Lenz G, Wright G, Dave SS, Xiao W, Powell J, Zhao H, et al. Stromal gene signatures in large-B-cell lymphomas. N Engl J Med. 2008 Nov 27;359(22):2313–23. doi: 10.1056/NEJMoa0802885 19038878

3. Alizadeh AA, Eisen MB, Davis RE, Ma C, Lossos IS, Rosenwald A, et al. Distinct types of diffuse large B-cell lymphoma identified by gene expression profiling. Nature. 2000 Feb 3;403(6769):503–11. doi: 10.1038/35000501 10676951

4. Hans CP. Confirmation of the molecular classification of diffuse large B-cell lymphoma by immunohistochemistry using a tissue microarray. Blood. 2004 Jan 1;103(1):275–82. doi: 10.1182/blood-2003-05-1545 14504078

5. Choi WWL, Weisenburger DD, Greiner TC, Piris MA, Banham AH, Delabie J, et al. A New Immunostain Algorithm Classifies Diffuse Large B-Cell Lymphoma into Molecular Subtypes with High Accuracy. Clinical Cancer Research. 2009 Aug 31;15(17):5494–502. doi: 10.1158/1078-0432.CCR-09-0113 19706817

6. Meyer PN, Fu K, Greiner TC, Smith LM, Delabie J, Gascoyne RD, et al. Immunohistochemical Methods for Predicting Cell of Origin and Survival in Patients With Diffuse Large B-Cell Lymphoma Treated With Rituximab. Journal of Clinical Oncology. 2011 Jan 6;29(2):200–7. doi: 10.1200/JCO.2010.30.0368 21135273

7. Gutierrez-Garcia G, Cardesa-Salzmann T, Climent F, Gonzalez-Barca E, Mercadal S, Mate JL, et al. Gene-expression profiling and not immunophenotypic algorithms predicts prognosis in patients with diffuse large B-cell lymphoma treated with immunochemotherapy. Blood. 2011 May 5;117(18):4836–43. doi: 10.1182/blood-2010-12-322362 21441466

8. Coupland SE. The challenge of the microenvironment in B-cell lymphomas. Histopathology. 2011 Jan 24;58(1):69–80. doi: 10.1111/j.1365-2559.2010.03706.x 21261684

9. Deeb SJ, D'Souza RCJ, Cox J, Schmidt-Supprian M, Mann M. Super-SILAC allows classification of diffuse large B-cell lymphoma subtypes by their protein expression profiles. Molecular & Cellular Proteomics. 2012 Feb 22;11(5):77–89. doi: 10.1074/mcp.M111.015362 22442255

10. Deeb SJ, Tyanova S, Hummel M, Schmidt-Supprian M, Cox J, Mann M. Machine Learning-based Classification of Diffuse Large B-cell Lymphoma Patients by Their Protein Expression Profiles. Molecular & Cellular Proteomics. 2015 Nov 1;14(11):2947–60.

11. Rüetschi U, Stenson M, Hasselblom S, Nilsson-Ehle H, Hansson U, Fagman H, et al. SILAC-Based Quantitative Proteomic Analysis of Diffuse Large B-Cell Lymphoma Patients. International Journal of Proteomics. 2015;2015:1–12.

12. Geiger T, Cox J, Ostasiewicz P, Wisniewski JR, Mann M. Super-SILAC mix for quantitative proteomics of human tumor tissue. Nature Methods. 2010 Apr 4;7(5):383–5. doi: 10.1038/nmeth.1446 20364148

13. Swerdlow SH, Campo EB, Harris NL, Jaffe ES, Pileri SA, Stein H, et al. WHO Classification of Tumours of Haematopoietic and Lymphoid Tissues. 4 ed. Vol. 2. 2017.

14. van der Meeren LE, Visser L, Diepstra A, Nijland M, van den Berg A, Kluin PM. Combined loss of HLA I and HLA II expression is more common in the non-GCB type of diffuse large B cell lymphoma. Histopathology. 2018;72(5):886–8. doi: 10.1111/his.13445 29194719

15. Ong S-E, Blagoev B, Kratchmarova I, Kristensen DB, Steen H, Pandey A, et al. Stable isotope labeling by amino acids in cell culture, SILAC, as a simple and accurate approach to expression proteomics. Mol Cell Proteomics. 2002 May;1(5):376–86. doi: 10.1074/mcp.m200025-mcp200 12118079

16. Ong S-E, Mann M. A practical recipe for stable isotope labeling by amino acids in cell culture (SILAC). Nat Protoc. 2007 Jan;1(6):2650–60.

17. Tang WH, Halpern BR, Shilov IV, Seymour SL, Keating SP, Loboda A, et al. Discovering Known and Unanticipated Protein Modifications Using MS/MS Database Searching. Anal Chem. 2005 Jul;77(13):3931–46. doi: 10.1021/ac0481046 15987094

18. Boeckmann B. The SWISS-PROT protein knowledgebase and its supplement TrEMBL in 2003. Nucleic Acids Research. 2003 Jan 1;31(1):365–70. doi: 10.1093/nar/gkg095 12520024

19. Meng X, Tackmann NR, Liu S, Yang J, Dong J, Wu C, et al. RPL23 Links Oncogenic RAS Signaling to p53-Mediated Tumor Suppression. Cancer Research. 2016 Aug 31;76(17):5030–9. doi: 10.1158/0008-5472.CAN-15-3420 27402081

20. Qi Y, Li X, Chang C, Xu F, He Q, Zhao Y, et al. Ribosomal protein L23 negatively regulates cellular apoptosis via the RPL23/Miz-1/c-Myc circuit in higher-risk myelodysplastic syndrome. Sci Rep. 2017 May 24;7(1):4841. doi: 10.1038/s41598-017-04711-8

21. Deeb SJ, D'Souza RCJ, Cox J, Schmidt-Supprian M, Mann M. Super-SILAC Allows Classification of Diffuse Large B-cell Lymphoma Subtypes by Their Protein Expression Profiles. Mol Cell Proteomics. 2012 May 14;11(5):77–89. doi: 10.1074/mcp.M111.015362 22442255

22. Huang X, Shen Y, Liu M, Bi C, Jiang C, Iqbal J, et al. Quantitative Proteomics Reveals that miR-155 Regulates the PI3K-AKT Pathway in Diffuse Large B-Cell Lymphoma. The American Journal of Pathology. 2012 Jul;181(1):26–33. doi: 10.1016/j.ajpath.2012.03.013 22609116

23. Visco C, Li Y, Xu-Monette ZY, Miranda RN, Green TM, Tzankov A, et al. Comprehensive gene expression profiling and immunohistochemical studies support application of immunophenotypic algorithm for molecular subtype classification in diffuse large B-cell lymphoma: a report from the International DLBCL Rituximab-CHOP Consortium Program Study. Nature Publishing Group; 2012 Aug 8;26(9):2103–13.

24. Staiger AM, Ziepert M, Horn H, Scott DW, Barth TFE, Bernd H-W, et al. Clinical Impact of the Cell-of-Origin Classification and the MYC/ BCL2Dual Expresser Status in Diffuse Large B-Cell Lymphoma Treated Within Prospective Clinical Trials of the German High-Grade Non-Hodgkin's Lymphoma Study Group. J Clin Oncol. 2017 Aug;35(22):2515–26. doi: 10.1200/JCO.2016.70.3660 28525305

25. Sarkozy C, Traverse-Glehen A, Coiffier B. Double-hit and double-protein-expression lymphomas: aggressive and refractory lymphomas. Lancet Oncology. 2015;16(15):e555–67. doi: 10.1016/S1470-2045(15)00005-4 26545844

26. Brouillard P, Boon LM, Mulliken JB, Enjolras O, Ghassibé M, Warman ML, et al. Mutations in a Novel Factor, Glomulin, Are Responsible for Glomuvenous Malformations (“Glomangiomas”). The American Journal of Human Genetics. 2002;70(4):866–74. doi: 10.1086/339492 11845407

27. Grisendi S, Chambraud B, Gout I, Comoglio PM, Crepaldi T. Ligand-regulated Binding of FAP68 to the Hepatocyte Growth Factor Receptor. J Biol Chem. 2001 Nov 30;276(49):46632–8. doi: 10.1074/jbc.M104323200 11571281

28. Tron AE, Arai T, Duda DM, Kuwabara H, Olszewski JL, Fujiwara Y, et al. The Glomuvenous Malformation Protein Glomulin Binds Rbx1 and Regulates Cullin RING Ligase-Mediated Turnover of Fbw7. Molecular Cell. Elsevier Inc; 2012 Apr 13;46(1):67–78.

29. Ruan J, Hyjek E, Kermani P, Christos PJ, Hooper AT, Coleman M, et al. Magnitude of Stromal Hemangiogenesis Correlates with Histologic Subtype of Non-Hodgkin's Lymphoma. Clinical Cancer Research. 2006 Oct 1;12(19):5622–31. doi: 10.1158/1078-0432.CCR-06-1204 17020964

30. Cardesa-Salzmann TM, Colomo L, Gutierrez G, Chan WC, Weisenburger D, Climent F, et al. High microvessel density determines a poor outcome in patients with diffuse large B-cell lymphoma treated with rituximab plus chemotherapy. Haematologica. 2011 Jun 30;96(7):996–1001. doi: 10.3324/haematol.2010.037408 21546504

Článek vyšel v časopise


2019 Číslo 10
Nejčtenější tento týden