Biomarker recommendation for PD‐1/PD‐L1 immunotherapy development in pediatric cancer based on digital image analysis of PD‐L1 and immune cells

Abstract

Anti-PD-1/PD-L1 immunotherapy could offer an alternative to traditional chemo-and/or radiotherapy to treat pediatric cancer patients. To unveil the potential benefit of this new therapeutic approach, the prevalence of PD-L1 and other relevant immune markers using quantitative digital image analysis (DIA) could help to clarify this point. A bridging study was first conducted using commercially available normal formalin-fixed paraffinembedded (FFPE) tonsils to compare immunostaining patterns and intensities from PD-L1, tumor infiltrating lymphocyte (TIL) markers CD3, CD8, FoxP3, CD45RO, and macrophage marker CD68 in adult (n = 5) and pediatric (n = 10) samples. Then, commercially available pediatric FFPE tumor samples from five prevalent pediatric solid tumor indications: ganglioneuroblastoma (n = 7); neuroblastoma (n = 23); nephroblastoma (n = 30); osteosarcoma (n = 24); and rhabdomyosarcoma (n = 25) were immunostained and their images (n = 654) digitally analyzed using predefined algorithms. The qualitative analysis of staining patterns and intensities in all 15 tonsils for all 6 biomarkers was similar regardless of age category. Quantitative DIA showed that PD-L1 values varied across cancer-types, nephroblastoma having the lowest counts. PD-L1 counts in ganglioneuroblastoma, our pediatric indication with the highest average value, was approximately 12-times lower than in a similar nonsmall cell lung cancer study, an indication approved for anti-PD-1/PD-L1 immunotherapies. Variable values were measured for the TIL markers CD3, CD8, and CD45RO. FoxP3 was scant across all indications. The macrophage marker CD68 showed highest values in ganglioneuroblastoma, with lowest levels in nephroblastoma. In conclusion, the low PD-L1 levels uncorrelated with TIL values from the present biomarker morphological study suggest that a PD-L1 immunohistochemistry patient selection strategy used for anti-PD-1/PD-L1 monotherapy in adult tumors may not succeed in these pediatric indications. Figure 5. Clustered heatmaps of the individual cases (y-axis) based on standardized expression levels of the individual markers across investigated indications. Red or blue indicates higher or lower than average levels, respectively. 132MA Silva et al

Systematic review of the immunological landscape of Wilms tumors

“…Silva et al. 42 confirmed that WT showed the lowest levels of PD-L1 expression within the group of pediatric solid tumors (ganglioneuroblastoma, neuroblastoma, osteosarcoma, rhabdomyosarcoma). Low PD-L1 expression correlated with low tumor-infiltrating lymphocytes.…”

“…With the exception of pediatric Hodgkin’s lymphoma and pediatric sarcomas, 37 PD-L1 expression was found to be generally low. 38 , 39 , 40 , 41 , 42 Routh et al. 40 analyzed 81 WT samples by immunohistochemistry: PD-L1 was expressed by only 14% of nephroblastomas and could be correlated with increased cancer recurrence.…”

Macrophages in Osteosarcoma Immune Microenvironment: Implications for Immunotherapy

“…Several studies have revealed that PD-L1 expression was observed in primary and metastatic tumors of osteosarcoma patients (46,137). PD-L1 positive tumors compared to PD-L1 negative tumors was significantly correlated with the presence of macrophages (137), particularly CD68 + cells (46,138), implicating the potential role of macrophages in the anti-PD1/ PD-L1 treatment. TAMs also expressed PD-1 to participate in immune escape and inhibit phagocytosis and anti-tumor immunity (139).…”

Clinical Relevance of CD4 Cytotoxic T Cells in High-Risk Neuroblastoma

“…Aoki et al first showed that neuroblastoma was PD-L1 negative (34). Later, others suggested that neuroblastoma cells per se expressed PD-L1 (35)(36)(37)(38). Recently, Shirinbak et al reported that PD-L1 protein was expressed predominately on tumor-associated macrophages (TAMs) infiltrating into neuroblastoma tissues at diagnosis and a very few neuroblastoma cells became PD-L1 positive after chemotherapy (39).…”