J. Hernandez

Integrating Molecular Testing in the Diagnosis and Management of Children with Thyroid Lesions

Thyroid nodules occur in 1–2% of children, and identifying which nodules are malignant is often challenging. Cytologic evaluation facilitates the diagnosis of thyroid lesions (TLs), but in 10–40% of cases the interpretation is indeterminate. Patients with indeterminate diagnoses are often treated with hemithyroidectomy followed by completion thyroidectomy, if cancer is found in the initial specimen. Exposing patients to multiple surgeries increases costs and morbidity. The American Thyroid Association states that a combination of molecular markers is likely to optimize the management of patients with indeterminate cytology. However, few studies have addressed the molecular alterations present in pediatric TL. Twenty-seven thyroid carcinomas from patients 10 to 19 years of age were tested for alterations common in adult TL, including BRAF V600E mutation, RET fusions, and TERT promoter mutations. Mutation-negative cases were subsequently analyzed with a next-generation sequencing (NGS) mutation panel to search for additional targets. Histologic diagnoses included 12 classic papillary thyroid carcinomas (PTCs), 13 follicular variant PTCs, 1 medullary thyroid carcinoma, and 1 follicular carcinoma. Fourteen cases showed lymph node involvement, and 13 cases demonstrated lymphovascular invasion. The BRAF V600E mutation was detected in 10/27 cases, and RET fusions were detected in 6/27 cases. No TERT promoter mutations were identified in any of the cases. The NGS panel revealed additional RET and CTNNB1 pathogenic missense mutations. Our results demonstrate that molecular abnormalities are common in pediatric TLs and suggest that incorporation of molecular testing will be helpful in optimizing patient management.

Abstract B24: Patient-derived tumor xenograft to study cancer stem cells is pediatric sarcomas

Tumor heterogeneity is recognized as an important hallmark in cancer. This characteristic allows cancer cells to evolve in time, to survive radiation and cyotoxic therapies, and to thrive in different organ microenvironments. Therefore, the presence of distinct tumor subpopulations, as mainstay feature of tumor heterogeneity, may play a role in tumor progression and treatment resistance. Patient-derived xenografts (PDXs) have been used to discover the molecular signaling pathways involved in tumorigenesis, metastasis formation and radiation/chemotherapy resistance in different epithelial-adult cancers. Our research interest in pediatric sarcomas is to understand the processes that drive tumor heterogeneity, to isolate and characterize cancer stem cells (CSCs), and to elucidate the mechanisms of chemoresistance using a PDX model. Pediatric patients with suspected or established diagnosis of sarcoma evaluated at Texas Children9s Hospital signed an informed consent to provide tumor samples for subcutaneous implantation of tumor explants in the flank of nonobese diabetic severe combined immunodeficient gamma (NSG) mice. Tumor explants were implanted on the same day of collection when was possible. First and subsequent PDX generations were harvested for tumor propagation, tumor histology, tumor cell karyotyping , RNA/DNA extraction for genetic studies and fluorescent activated cell sorting (FACS) analysis for ALDH, CD133, EphA2 and CD47. Forty-six tumor samples have been collected since June 2013. Half of tumor samples (n=21) corresponded to osteosarcoma patients and 59% of all tumor specimens were obtained at the time of the initial biopsy. Approximately 66% of biopsies have been performed by Interventional Radiology in our institution. All PDXs maintained the same histological characteristics that original tumors, and also presented specific genetic translocations associated with the different sarcoma types. Chemotherapy resistant and very aggressive tumors (metastatic) had short tumor latency and generate tumors in all transplanted mice. We did not observe the formation of metastases (lungs or liver) in any of the transplanted PDX mice. PDXs significantly increased the amount of tumor material necessary to perform cell culture under stem-like cell conditions and to analyze the expression of CSC markers by FACS. These studies would be very difficult to perform directly from patient tumor biopsies. All tumors harvested (n=15) contained 2-9% of ALDH Hi or stem-like cells. None of the PDXs analyzed expressed CD133 but synovial sarcoma. We found that >90% of sarcoma cells expressed CD47 and EphA2 at different intensity levels. Whole genome sequencing of different tumor cell subpopulations and limiting dilution experiments to validate the presence of CSCs are ongoing. Core needle biopsies would represent the most common source to obtain tumor specimens before treatment in pediatric sarcomas. The development of PDXs allows the propagation of tumor cell subpopulations, including CSCs, without the biological changes impose by cell culture. PDXs resemble the original tumor and constitute a powerful tool to study tumor heterogeneity. We plan to perform orthotopic transplantation and tail vein injection of PDX cells in an attempt to develop a PDX model of metastatic disease in pediatric sarcomas. Citation Format: Nino Rainusso, Jose Hernandez, Rex Marco, Sanjeev Vasudevan, Norma Quintanilla, John Hicks, Wendy Allen-Rhoades, Matteo Trucco, Jason Yustein. Patient-derived tumor xenograft to study cancer stem cells is pediatric sarcomas. [abstract]. In: Proceedings of the AACR Special Conference: Patient-Derived Cancer Models: Present and Future Applications from Basic Science to the Clinic; Feb 11-14, 2016; New Orleans, LA. Philadelphia (PA): AACR; Clin Cancer Res 2016;22(16_Suppl):Abstract nr B24.