Plasma Affinity Proteomic Immunoprofiling As a Novel Prognostic Tool in High Risk Diffuse Large B-Cell Lymphoma Patients: A Nordic Lymphoma Group Study

Background

Despite recent advances in molecular profiling of diffuse large B-cell lymphoma (DLBCL), only a few biomarkers currently have an impact on treatment. In a previous study we could observe the heterogeneity of DLBCL in the plasma immunoprofile from DLBCL patients, by use of recombinant antibody microarrays. By unsupervised hierarchical clustering, an immunoprofile of 23 plasma proteins could divide patients into two subgroups with significantly different overall survival (OS). In this study we aimed to expand the immunoprofiling with longitudinal plasma samples from high risk DLBCL patients, to search for novel prognostic and potentially predictive markers.

Material and Methods

Plasma samples from 126 high risk DLBCL patients included in a phase II clinical trial of the Nordic Lymphoma Group (CRY04) were collected at diagnosis, during and after treatment, and in the event of progression or relapse. Inclusion criteria were age 18-65 years, primary diffuse large B-cell lymphoma without CNS involvement, age-adjusted International Prognostic Index (aaIPI) 2-3 and WHO performance score 0-3. Six courses of R-CHOEP-14 were given followed by systemic CNS prophylaxis with one course of high-dose cytarabine and one course of high-dose methotrexate. Additionally, age and sex matched controls (n = 40) were included. Two hundred and eighty-three recombinant scFv antibody fragments directed against 97 known serum antigens, mainly immunoregulatory proteins, were selected from phage display libraries to be used in the antibody microarrays.

Results

Immunoprofiles distinguishing DLBCL patients from healthy controls were identified. Furthermore, a different protein expression was found in patients with aaIPI 3 versus 2 as well as in patients with shorter versus longer progression free survival (PFS). The kinases CDK-2 and BTK were upregulated both in patients with higher aaIPI score and in patients with shorter PFS. Comparing the patients who developed disease progression with those who did not, seven differentially deregulated proteins were identified including the phosphatase PTP-1B, the chemokine MCP-1, and the cytokines IL-4, IL-6 and IL-12, all previously implicated in B-cell lymphoma pathogenesis. In addition, results showed that subdivision of patients according to immunoprofile as defined in our previous study, could be performed also in the present patient cohort. The immunoprofile comprises T-helper (TH)1 cytokines, TH2 cytokines, complement proteins, chemokines, enzymes, and membrane proteins. The 23 included proteins are β-galactosidase, C1 esterase inhibitor, C4, C5, Cystatin C, Eotaxin, GLP-1 R, GM-CSF, HLA-DR/DP, IgM, IL-1ra, IL- 2, IL- 3, IL- 6, IL- 10, IL- 12, Leptin, MCP-1, MCP-3, Mucin-1, PSA, TNF-α, and TNF-β. Although not significant, a potential association to PFS and OS could be observed between the two generated subgroups. Finally, expression of IL-10 was shown to improve the prognostic value of aaIPI regarding both PFS and OS.

Conclusion

Protein expression profiling of plasma from high risk DLBCL patients provided novel insights into the biology of DLBCL. New candidate prognostic markers were identified which could potentially guide us in the prediction of outcome and in the choice of treatment for DLBCL patients. However, as this study was aimed for discovery, the findings must be validated in future studies with independent patient cohorts.