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

Abstract

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.

Proteomic analysis in diffuse large B-cell lymphoma identifies dysregulated tumor microenvironment proteins in non-GCB/ABC subtype patients

“…In a recent proteomic study [12], which also could separate ABC and GCB patients by their global protein profile in FFPE samples (n ¼ 42), we could significantly reproduce 6 of 8 proteins in their separating 8-protein signature. Three other smaller studies have also performed proteomic COO analyses in DLBCL patients [9][10][11]; however, they could not reproduce well-known COO proteins and even if there was some minor overlap, none of their top-upregulated proteins in the different subgroups were upregulated in our study. In summary, we believe that the reliability of our protein identification and expression patterns is high, judging by the overlap both of the proteins identified by the Deeb and Reinders studies and most of the corresponding genes separating the ABC subtype from GCB in the Lymph2cx gene chip, as well as a reproduction of some of the differentially regulated proteins using immunohistochemical staining.…”

“…Instead, methods to directly study the global protein expression and interaction could bring new knowledge regarding the pathophysiology of DLBCL subgroups. Some global protein expression studies using a proteomic approach where non-GCB or ABC patients are compared to controls or their GCB counterpart have been published [8][9][10][11][12]. Even though these studies show promising results, the results have been diverging which at least partly could be explained by the use of different techniques and perhaps foremost the low total number of included patients (ranging from a total of 6-42 patients).…”

Characterization of Human B Cell Hematological Malignancies Using Protein-Based Approaches

“…For a deeper characterization, LC-MS strategies are commonly used. Thus, van der Meeren et al [87] profiled the two subtypes of DLBCL (GCB and non-GCB), analyzing more than 4200 proteins of which 37 were found significantly differentially expressed between the two subtypes. Validation of these markers revealed that the expression of glomulin protein was higher in GCB-DLBCL patients, whereas ribosomal protein L23 was a biomarker for non-GCB-DLBCL individuals.…”

Meta-Analysis of MS-Based Proteomics Studies Indicates Interferon Regulatory Factor 4 and Nucleobindin1 as Potential Prognostic and Drug Resistance Biomarkers in Diffuse Large B Cell Lymphoma

“…(2) bias due to deviations from the intended intervention; (3) bias due to missing outcome data; (4) bias in the measurement of the outcome; and (5) bias in the selection of the reported results (Figure S3A,B). The results of the risk of bias assessment revealed one study with a high risk [48], seven with a low risk [27,[49][50][51][52][53][54], and nineteen with a moderate risk of bias [26,[55][56][57][58][59][60][61][62][63][64][65][66][67][68][69][70][71][72].…”