Immuno-transcriptomic profiling of extracranial pediatric solid malignancies

Abstract

We perform an immunogenomics analysis utilizing whole-transcriptome sequencing of 657 pediatric extracranial solid cancer samples representing 14 diagnoses, and additionally utilize transcriptomes of 131 pediatric cancer cell lines and 147 normal tissue samples for comparison. We describe patterns of infiltrating immune cells, T cell receptor (TCR) clonal expansion, and translationally relevant immune checkpoints. We find that tumor-infiltrating lymphocytes and TCR counts vary widely across cancer types and within each diagnosis, and notably are significantly predictive of survival in osteosarcoma patients. We identify potential cancer-specific immunotherapeutic targets for adoptive cell therapies including cell-surface proteins, tumor germline antigens, and lineage-specific transcription factors. Using an orthogonal immunopeptidomics approach, we find several potential immunotherapeutic targets in osteosarcoma and Ewing sarcoma and validated PRAME as a bona fide multi-pediatric cancer target. Importantly, this work provides a critical framework for immune targeting of extracranial solid tumors using parallel immuno-transcriptomic and -peptidomic approaches.

Keywords: PRAME; RNA sequencing; T cell receptor; adoptive cell therapy; immunogenomics; immunopeptidomics; pediatric oncology; tumor-infiltrating lymphocytes.

Figure 1.. Tumor immune microenvironment of pediatric solid tumors

(A) Global pattern of enrichment of various immune signatures across cancer types. Tumor types with a sample size of >5 are shown. The heatmap corresponds to the percentage of tumors with a positive enrichment score for the immune cell subtype by ssGSEA. (B) Distribution of immune signature enrichment scores across cancer types included in this study (colored) as compared to adult tumor samples in the TCGA project (gray). (C and D) Kaplan-Meier (KM) plots of overall survival demonstrate that patients with tumors of high immune score (C) or high CD8⁺ T cell score (D) are significantly associated with a favorable prognosis in the osteosarcoma cohort where outcome data are available.

Figure 2.. Immune checkpoint expression in pediatric solid tumors

(A) Expression of selected immune checkpoint genes across tumor types. Dots represent the median expression for each cancer type. (B) Correlation of immunomodulatory gene expression and CD8⁺ T cell infiltrate. Fill indicates significant association (Spearman rank correlation >0.3; adjusted p < 0.05) within that cancer type. Highlighted genes in the blue font represent targets of antibody therapies approved by FDA or currently in clinical trial. (C) Protein expression of an immune gene panel on an independent neuroblastoma tissue array (Wei et al., 2018) using a multiplex protein detection assay reveal consistent findings of differential expression of immune cell markers between MYCN-amplified (MYCN.A) and MYCN-not amplified (MYCN.NA) tumors. The scale bar represents Z-scored standardized protein-expression level.

Figure 3.. Intra-tumoral T cell receptor β (TCR-β) repertoire identified using RNA-seq data in pediatric solid tumors

(A) Number of unique complementary-determining region 3 (CDR3) detected in each tumor. Red bars represent median for each cancer type. (B) Kaplan-Meier analysis of available outcome data in the osteosarcoma cohort demonstrates that patients with a high TCR-β count are significantly associated with favorable outcome (p < 0.01). (C) In order to investigate T cell clone expansion in individual tumors, TCR-β clones are ranked by their abundance on the x axis and the normalized clone count is plotted on the y axis. Each line represents all TCR-β clones detected in a single tumor and clearly shows evidence of high clonal expansion of some TCRs. (D) Clonal expansion of TCR-βs. Each dot represents a TCR-β clone in a tumor sample. The highlighted region depicts expanded TCR-β clones as evidenced by high normalized clone count (>99^th percentile) and high relative contribution to the total intra-tumoral TCR-β count (>1%). The accompanying table details the percentage of tumors with ≥1 clonally expanded TCR-β. *Total tumor/patient count and calculated percentages include all patients in the study cohort.

Figure 4.. Tumor-specific gene expression

(A–C) Tumor-specific gene expression including (A) cell-surface proteins, (B) transcription factors, and (C) tumor germline antigens. Fill indicates that the gene is overexpressed in the corresponding cancer type relative to normal tissues and has minimal expression in vital organs. (D) mRNA expression of top genes for each category in each cancer type, vital organs, testes, ovary, and other normal tissues. Dots represent the median expression for each cancer type. (E) Representative PRAME immunohistochemistry in Ewing sarcoma and osteosarcoma demonstrates a robust expression of PRAME protein in tumor cells. H&E, hematoxylin and eosin stain.

Figure 5.. Specific anti-tumor activity of engineered T cells targeting a PRAME MHC class 1 peptide

(A) Structure of engineered PRAME TCR and schema for testing the specificity and efficacy of murPRAME-TCR T cells. (B) In vitro co-culture of T cells with reporter cell lines at different effector:tumor (E:T) ratios. Luminescence was measured after 24 h of co-culture and reported as mean ± SEM (n = 3); **p < 0.01, ***p < 0.001. (C) Schema for treating metastatic EWS xenograft model with murPRAME-TCR T cells. (D) Bioluminescence images of TC32-Luc:PRAME cells after IV injection and treatment with vehicle or T cells. (E) Quantification of bioluminescence imagining reported as mean ± SEM (n = 8 per group). p values of UTD versus murPRAME-TCR mice displayed as *p < 0.05, **p < 0.01. (F) Kaplan-Meier analysis of mouse survival using log-rank test (n = 8 per group).