PILOT ASSESSMENT OF PROXIMITY EXTENTION IMMUNOASSAY IN PATIENTS WITH MONOCLONAL GAMMOPATHIES: PARALLEL EVALUATION OF 92 CANCER-RELATED PARAMETERS IN BONE MARROW AND IN SERA

Background
The assessment of prognostic factors in cancer is vital for correct stratification and targeted therapy. In multiple myeloma, both tumor cells and bone marrow microenvironment play an important role in pathogenesis, and take part on the development and the extent of myeloma bone disease (MBD).

Aims
The aim of our project was to address the utility of Proximity Extention ImmunoAssay technique (PEA) in patients with monoclonal gammopathies instead of standard ELISA techniques in the assessment of tumor microenvironment parameters.

Methods
We assessed 92 cancer-related parameters from the Oncology I ^96x96 protein biomarker panel (Olink, Uppsala, Sweden). The parameters are known to be altered in various cancer types and are related to angiogenesis, cell-cell signaling, growth control and inflammation. Both sera and bone marrow samples were acquired from 30 patients with monoclonal gammopathies - 25 patients with multiple myeloma (MM) and 5 individuals with monoclonal gammopathy of undetermined significance (MGUS) taken at the time of diagnosis.PEA is a sensitive and specific alternative of ELISA methods. For each sample there are two different antibody probes labeled with A- and B-oligonucleotides. Proximity of the oligonucleotide chains to the binding site leads to PCR amplification of the signal with specific primers. For statistical estimation we used Mann-Whitney U-test, Kruskal-Wallis test and Spearman correlation analysis at p?0.05 and Principal Component Analysis (GenEx, SSPS).

Results
In 3 patients the data did not pass the quality control. In the rest 27 individuals we analysed parallelly bone marrow and sera analytes. In 58 analytes the levels in bone marrow and serum were significantly different, with up to 38 fold difference (over 5 log scale), the rest 34 analytes had similar levels in both bone marrow and sera. Out of the whole panel, 9 analytes were significally different when comparing individuals with or without the presence of renal impairment. We found 19 proteins that significantly differed between MGUS and MM in both serum and bone marrow. There were several perspective candidate biomarkers correlating with disease stage, extent of myeloma bone disease, M-protein type and therapeutic outcome, such as tartrate resistant alkalinephosphatase-5 (TRAP-5), matrix metalloproteinase 1 (MMP-1), tumor necrosis factor (TNF), transforming growth factor alpha (TGF-α), Fas antigen ligand (FasL), Fms-related tyrosine kinase 3 ligand (Flt3-L) or epithelial cell-adhesion molecule (Ep-CAM).

Summary
Proximity ImmunoAssay is a very sensitive technique enabling parallel assessment of several analytes. Unlike ELISA, it has many fold larger dynamic range, and is suitable for analyte levels beyond the calibration curve. Out of the Oncology panel, 9 parameters were influenced by renal impairment. Despite limited number of patients, we could trace several potential candidates for the assessment of  MM pathogenesis as well as for the extent of myeloma bone disease.

Supported by the grant NT14393

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