Computational protocol: Expression and clinical association of programmed cell death-1, programmed death-ligand-1 and CD8+ lymphocytes in primary sarcomas is subtype dependent

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Protocol publication

[…] Statistical analyses were performed using IBM SPSS Statistics 22 and p-value < 0.05 was considered significant and p-value < 0.1 was considered a trend. Relations between categorical parameters were assessed by Chi-square or Fisher’s exact testing and associations with overall (OS), event-free (EFS) and metastases-free survival (MFS) were assessed by the Kaplan-Meier method with Log rank test. Tumors positive for one marker or a combination were compared to tumors negative for that particular marker or combination. A distinction between the different levels of expression (< 10, 10–50, ≥ 50) was made in the analysis. [...] PD-1 expression was analyzed by flow cytometry: 1 × 105 cells per cell line were washed in PBS with 2% FBS (Gibco) and 0.5% BSA (Sigma) (PBA) followed by 20 min incubation at RT with either mouse anti-human PD-1 (CD279) antibody (BB515, 1:20 in PBA, BD Horizon) or mouse IgG1 κ isotype antibody (BB515 Clone X40, 1:80 in PBA, BD Horizon). CHO cell line transfected with a pcDNA3.1+/C-(K)DYK vector enriched with PDCD1 for 48h acted as a positive control (Lipofectamine 3000 Transfection kit™, Thermo Scientific). Cells were washed and dissolved in PBA followed by flow cytometry using FACS Verse (BD Biosciences). Data analysis was performed using FLowJo version 10.0.7. […]

Pipeline specifications

Software tools SPSS, FlowJo
Applications Miscellaneous, Flow cytometry
Organisms Homo sapiens