Andrew L. Kung

Purpose: Molecular diagnostics are standard of care for most adults with solid malignancies, but implementation in pediatric solid tumors remains inconsistent. The SPROUT (Somatic Profiling for Pediatric Cancer Refining our Understanding and Treatment) Molecular Guidelines Working Group was convened to develop molecular testing guidelines for pediatric solid tumors. Experimental Design: A structured literature review was performed using Covidence to identify relevant clinical studies evaluating molecular diagnostics in pediatric solid tumors. The literature and collective clinical experience of the multidisciplinary working group were used to develop consensus recommendations for molecular profiling at diagnosis and at relapse or recurrence. Results: Of 361 manuscripts screened, 41 studies were included for data extraction. Based on the available evidence and expert consensus, recommendations were developed for the use and timing of DNA and RNA next-generation sequencing and methylation profiling across pediatric solid tumors. Conclusions: The SPROUT Molecular Guidelines provide a framework for standardized implementation of molecular diagnostics in pediatric solid tumors. Broad adoption of these recommendations may improve diagnostic precision, reduce variability in molecular testing, and increase opportunities for biomarker-directed therapy and clinical trial enrollment for children with cancer.

Supplementary Figure 1. Activation status of anti-U5 snRNP200 CAR T cells co-cultured with AML cells in vitro. Supplementary Figure 2. Efficacy of IL-18 armored anti-U5 snRNP200 CAR T cells and their dependency on IFNg secretion. Supplementary Figure 3. Efficacy of IL-18 armored anti-U5 snRNP200 CAR T cells in AMLPDX models. Supplementary Figure 4. Evaluation of IL-18 armored U5 snRNP200 CAR T cells in pediatric AML PDXs. Supplementary Figure 5. Safety assessment of anti-U5 snRNP200 CAR T cells. Supplementary Figure 6. Efficacy of mouse IL-18 secreting U5 snRNP200 CAR T cells in a syngeneic mouse AML model. Supplementary Figure 7. UMAPs of bone marrow cells annotated by cell type and marker gene expression in cell type. Supplementary Figure 8. Clonal expansion of endogenous T cells in mice treated with IL18 secreting anti-U5 snRNP200 CAR T cells. Supplementary Figure 9. Evaluation of mechanism underlying AML re-challenge in immunocompetent mice following AML treatment with U5 snRNP200 CAR T cells. Supplementary Figure 10. Surface expression of U5 snRNP200 and efficacy of anti-U5 snRNP200 CAR T cells in B-ALL. Supplementary Figure 11. Anti-leukemic activity of U5 snRNP200 against B-ALL in vivo.

Supplementary Table S1. Clinical and genetic characteristics of AML and B-ALL patients for PDX models. Supplementary Table S2. TCR sequences of bone marrow T cells in syngeneic AML mouse model. Supplementary Table S3. Clinical and genetic characteristics of B-ALL patients for evaluation of surface U5 snRNP200 expression.

Preclinical translational research has increasingly utilized patient-derived xenograft (PDX) models for mechanistic and experimental therapeutic studies, yet most existing models have been developed from adult cancer types. We describe the establishment of a PDX program to expand the availability of pediatric-specific PDXs for preclinical research and enable studies of pediatric cancer histologies, including ultrarare diseases. Processes for PDX generation were integrated into established clinical workflows to facilitate universal model generation. Methodologies for tissue procurement, processing, and cryopreservation were optimized to enable intra- and interinstitutional PDX model generation. Over a 6-year span, 388 PDX tumor models representing more than 40 diagnoses were generated, including ultrarare tumors and longitudinal models established from pretherapy, posttherapy, and relapse tumors from the same patient. Genomic characterization of these PDXs demonstrates excellent concordance and recapitulation of molecular alterations of the source tumor. Successful PDX generation was enhanced from relapsed samples, was higher in sarcomas compared with other solid tumor types, and was a negative prognosticator for clinical outcome. With a broad portfolio of molecularly annotated models, we demonstrate utility for validating cross-histology biomarker-driven therapeutic strategies by demonstrating antitumor activity of an MAT2A inhibitor in MTAP-deficient PDXs. Universal model creation also allows for experimental validation of therapeutic hypotheses on a patient-specific basis, as we describe the characterization of a novel RAF1 fusion (EPB41L2::RAF1) in an osteosarcoma PDX. Development of a diverse collection of pediatric PDX models enables hypothesis-driven and cross-histology studies that expand our understanding of cancer biology and aid ongoing drug prioritization efforts in rare tumors. Significance:A clinically integrated, genomically annotated pediatric PDX portfolio supported by systematic benchmarking of model generation facilitates exploratory biomarker-driven and patient-specific translational studies.

Optimization efforts to procure patient tumors, establish PDX tumor models and cryopreserve PDX tumor specimen.

Comparative analysis of mutations in patient source tumors and corresponding PDX models.

Validation of a EPB41L2::RAF1 gene fusion in an osteosarcoma PDX model.

Evaluation of the EPB41L2::RAF1 gene fusion as an oncogenic driver and therapeutic target in osteosarcoma PDX models.

Unsupervised hierarchical clustering of sarcoma patient tumors and corresponding PDX models.

Unsupervised hierarchical clustering of osteosarcoma patient tumors and corresponding PDX models.