br Laboratory measurements br The SOMAscan array
2.3. Laboratory measurements
The SOMAscan array is a commercially available assay from the com-pany SomaLogic (Boulder, CO USA). We sent 35 serum samples taken from SHOC patients at remission to SomaLogic. After the SOMAscan array analysis was complete, the data was sent back, which was then analyzed to determine, which proteins had the best hazard ratios and p-value for predicting time to death on univariate analysis . The pro-teins with a log rank p-value of b0.05 and hazard ratio N2.5 were se-lected for in house confirmation via multiplex Luminex assay. Multivariate analysis was not performed on the significant SOMAscan
proteins. The majority of significant proteins on the SOMAscan array did not have a corresponding Luminex assay available and were omitted from Luminex analysis.
2.4. Validation of SOMAScan data by Luminex assay
We elected to measure twenty-six proteins based on the SOMAscan array results as well as previously published data . These proteins were Insulin-like Growth Factor Binding Proteins (IGFBP1, 2, 5, 6 and 7,) Interferon Gamma (IFNγ), interleukins (IL4, IL6, sIL6R, IL10, IL13, IL15), Macrophage Inflammatory Proteins (CCL3 and CCL4), Monocyte Chemotactic Protein 1 (MCP1/CCL2, MCP3/CCL7), Macrophage Derived Chemokine (MDC/CCL22), tissue Plasminogen activator inhibitor-1 (tPAI1), Platelet Derived Growth Factors (PDGF.AA and PDGF.ABBB), Regulated on Activation, Normal T cell Expressed and Secreted (RANTES/CCL5), soluble Glycoprotein 130 (sgp130), soluble Intercellu-lar Adhesion Molecule 1 (sICAM1), soluble Tumor Necrosis Factor Re-ceptors (sTNFRII and, sTNFRI), and soluble Vascular Adhesion Protein 1 (sVCAM1) . These proteins were examined for their ability to pre-dict PFS when drawn at remission.
Luminex assays for the above mentioned 26 proteins were obtained from Millipore (Millipore Inc., Billerica, MA, USA). Multiplex assays were performed according to instructions provided with the kit. Serum samples were incubated with capture Epirubicin HCl immobilized on polystyrene beads for 1 h. The beads were then washed and further incubated with biotinylated detection antibody cocktail for 1 h. Next, beads were washed twice to remove unbound detection antibody, and then incubated with phycoerythrin-labeled streptavidin for 30 min. Last, beads were washed and suspended in 60 μl of wash buffer.
The median fluorescence intensities (MFI) were measured using a FlexMAP 3D array reader (Millipore, Billerica, MA) with the following instrument settings: events/bead: 50, minimum events: 0, flow rate: 60 μl/min, Sample size: 50ul and discriminator gate: 8000–13,500.
Before performing the profiling, assays were performed at different serum dilutions to ensure the MFI values of the samples were within the linear range of the standard curve.
Luminex median fluorescence intensity (MFI) data was subjected to quality control steps as described in our earlier study . Described briefly, wells with low bead counts (below 30), or high bead CV (above 200) were flagged for exclusion. The coefficient of variation of replicate wells was also checked and wells with CV N 25% were not in-cluded in further analyses.
Protein concentrations were estimated using a regression fit to the standard curve with known concentration included on each plate using a serial dilution series. To achieve normal distribution, MFI and concentrations for standards were log2 transformed prior to all statisti-cal analyses.
2.5. Statistical analysis
All statistical analyses were performed using the R language and en-vironment for statistical computing (RStudio version 1.1.383; R Founda-tion for Statistical Computing; www.r-project.org). The protein concentrations were log2 normalized after initial QC. The statistical sig-nificance of differences was set at p b 0.05, all p values were two sided. Correlation analysis between serum protein concentrations were per-formed using the Pearson parametric correlation test. Cox proportional hazards models were used to evaluate the impact of clinical factors and serum protein levels on PFS. These results are reported with corre-sponding 95% confidence intervals. Patients with no history of recur-rence or death were censored at the date of last follow-up visit. Patients principle of independent assortment died of natural causes unrelated to cancer were censored at time of death. Patient PFS was censored at 10 years. Kaplan-Meier survival analysis and log-rank test were used to compare differences in PFS between 10 groups classified based on each 10th percentile of