Background: Significant anti-tumor effects have been observed in a variety of malignancies via blockade of immune checkpoints, leading to T-cell-mediated killing of cancer cells. Interaction of PD-1 with its ligands PD-L1 and PD-L2 serves to suppress T-cell function and restrict immune-mediated tumor killing. We sought to examine the pattern of expression of these proteins in children with neuroblastoma, as expression of these proteins may serve as biomarkers of response.
Methods: Sections cut from formalin fixed paraffin embedded (FFPE) tissue blocks were processed and evaluated for PD-1, PD-L1, and PD-L2 by immunohistochemistry (IHC) as well as by mRNA expression. A semi-quantitative 0-5 IHC scoring system (0= negative, 1 = rare, 2 = low, 3 = moderate, 4 = high, 5 = very high) was applied, with scores incorporating combined prevalence of tumor cell and non-tumor cell labeling. Expression profiling was performed using the NanoString nCounter™ system according to manufacturers’ recommendations. Data analysis was performed using quantile normalization in which relative ranks of genes (across all genes on the Nanostring codeset) within each sample were replaced by values having the same relative rank from the pooled distribution (from all samples and genes in the dataset). All quantile normalized data underwent subsequent log10 transformation.
Results: 31 FFPE blocks were included in the analysis. PD-1 and PD-L1 IHC were evaluable in all samples. 6/31 samples were not evaluable for PD-L2 IHC. PD-1, PD-L1 and PD-L2 expression was negative to moderate by both IHC (range 0-3) and mRNA expression (range 0-2.19). Correlation between IHC score and mRNA expression was poor for all three tested proteins (PD-1 r2 = 0.07, PD-L1 r2 = 0.1 and PD-L2 r2 = 0.1).
Conclusions: Expression of PD-1, PD-L1 and PD-L2 is low in neuroblastoma. At low levels of expression, IHC score and mRNA expression correlate poorly.