University of Chicago Hospital, Chicago, IL
Navin R. Pinto , Julie R. Park , Erin Murphy , Jennifer Yearley , Terri McClanahan , Lakshmanan Annamalai , Douglas S. Hawkins , Erin R. Rudzinski
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 solid tumors [Ewing sarcoma(ES), neuroblastoma(NB), osteosarcoma(OS), Wilms tumor(WT) and rhabdomyosarcoma(RM)], 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: 124 FFPE blocks were included in the analysis (ES = 20, NB = 31, OS = 25, WT = 25, RMS = 23) . PD-1, PD-L1 and PD-L2 IHC were not evaluable in 8, 0 and 12 blocks, respectively. PD-1, PDL-1 and PDL-2 expression was negative to moderate by both IHC (range 0-3.5) and mRNA expression (range 0-2.62). Correlation between IHC score and mRNA expression was poor for all three tested proteins (PD-1 r2 = 0.06, PDL-1 r2 = 0.007 and PDL-2 r2= 0.15). Conclusions: Expression of PD-1, PD-L1 and PD-L2 is low in pediatric solid tumors. At low levels of expression, IHC score and mRNA expression correlate poorly.
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