Claire Sinai

Clinical targeted exome-based sequencing in combination with genome-wide copy number profiling: precision medicine analysis of 203 pediatric brain tumors

Background Clinical genomics platforms are needed to identify targetable alterations, but implementation of these technologies and best practices in routine clinical pediatric oncology practice are not yet well established. Methods Profile is an institution-wide prospective clinical research initiative that uses targeted sequencing to identify targetable alterations in tumors. OncoPanel, a multiplexed targeted exome-sequencing platform that includes 300 cancer-causing genes, was used to assess single nucleotide variants and rearrangements/indels. Alterations were annotated (Tiers 1-4) based on clinical significance, with Tier 1 alterations having well-established clinical utility. OncoCopy, a clinical genome-wide array comparative genomic hybridization (aCGH) assay, was also performed to evaluate copy number alterations and better define rearrangement breakpoints. Results Cancer genomes of 203 pediatric brain tumors were profiled across histological subtypes, including 117 samples analyzed by OncoPanel, 146 by OncoCopy, and 60 tumors subjected to both methodologies. OncoPanel revealed clinically relevant alterations in 56% of patients (44 cancer mutations and 20 rearrangements), including BRAF alterations that directed the use of targeted inhibitors. Rearrangements in MYB-QKI, MYBL1, BRAF, and FGFR1 were also detected. Furthermore, while copy number profiles differed across histologies, the combined use of OncoPanel and OncoCopy identified subgroup-specific alterations in 89% (17/19) of medulloblastomas. Conclusion The combination of OncoPanel and OncoCopy multiplex genomic assays can identify critical diagnostic, prognostic, and treatment-relevant alterations and represents an effective precision medicine approach for clinical evaluation of pediatric brain tumors.

Rethinking childhood ependymoma: a retrospective, multi-center analysis reveals poor long-term overall survival

Ependymoma is the third most common brain tumor in children, but there is a paucity of large studies with more than 10 years of follow-up examining the long-term survival and recurrence patterns of this disease. We conducted a retrospective chart review of 103 pediatric patients with WHO Grades II/III intracranial ependymoma, who were treated at Dana-Farber/Boston Children’s Cancer and Blood Disorders Center and Chicago’s Ann & Robert H. Lurie Children’s Hospital between 1985 and 2008, and an additional 360 ependymoma patients identified from the Surveillance Epidemiology and End Results (SEER) database. For the institutional cohort, we evaluated clinical and histopathological prognostic factors of overall survival (OS) and progression-free survival (PFS) using the log-rank test, and univariate and multivariate Cox proportional-hazards models. Overall survival rates were compared to those of the SEER cohort. Median follow-up time was 11 years. Ten-year OS and PFS were 50 ± 5% and 29 ± 5%, respectively. Findings were validated in the independent SEER cohort, with 10-year OS rates of 52 ± 3%. GTR and grade II pathology were associated with significantly improved OS. However, GTR was not curative for all children. Ten-year OS for patients treated with a GTR was 61 ± 7% and PFS was 36 ± 6%. Pathological examination confirmed most recurrent tumors to be ependymoma, and 74% occurred at the primary tumor site. Current treatment paradigms are not sufficient to provide long-term cure for children with ependymoma. Our findings highlight the urgent need to develop novel treatment approaches for this devastating disease.

Developmental and oncogenic programs in H3K27M gliomas dissected by single-cell RNA-seq

The cellular composition of H3K27M gliomas Diffuse midline gliomas with histone H3 lysine27-to-methionine mutations (H3K27M-glioma) are an aggressive type of childhood cancer with few options for treatment. Filbin et al. used a single-cell sequencing approach to study the oncogenic programs, genetics, and cellular hierarchies of H3K27M-glioma. Tumors were mainly composed of cells resembling oligodendrocyte precursor cells, whereas differentiated malignant cells were a smaller fraction. In comparison with other gliomas, these cancers had distinct oncogenic programs and stem cell–like profiles that contributed to their stable tumor-propagating potential. The analysis also identified a lineage-specific marker that may be useful in developing therapies. Science, this issue p. 331 Single-cell analyses of H3K27M glioma defines a putative developmental hierarchy that differs from other gliomas. Gliomas with histone H3 lysine27-to-methionine mutations (H3K27M-glioma) arise primarily in the midline of the central nervous system of young children, suggesting a cooperation between genetics and cellular context in tumorigenesis. Although the genetics of H3K27M-glioma are well characterized, their cellular architecture remains uncharted. We performed single-cell RNA sequencing in 3321 cells from six primary H3K27M-glioma and matched models. We found that H3K27M-glioma primarily contain cells that resemble oligodendrocyte precursor cells (OPC-like), whereas more differentiated malignant cells are a minority. OPC-like cells exhibit greater proliferation and tumor-propagating potential than their more differentiated counterparts and are at least in part sustained by PDGFRA signaling. Our study characterizes oncogenic and developmental programs in H3K27M-glioma at single-cell resolution and across genetic subclones, suggesting potential therapeutic targets in this disease.

Immunophenotyping of pediatric brain tumors: correlating immune infiltrate with histology, mutational load, and survival and assessing clonal T cell response

There is little known regarding the immune infiltrate present in pediatric brain tumors and how this compares to what is known about histologically similar adult tumors and its correlation with survival. Here, we provide a descriptive analysis of the immune infiltrate of 22 fresh pediatric brain tumor tissue samples of mixed diagnoses and 40 peripheral blood samples. Samples were analyzed using a flow cytometry panel containing markers for immune cell subtypes, costimulatory markers, inhibitory signals, and markers of activation. This was compared to the standard method of immunohistochemistry (IHC) for immune markers for 89 primary pediatric brain tumors. Both flow cytometry and IHC data did not correlate with the grade of tumor or mutational load and IHC data was not significantly associated with survival for either low grade or high grade gliomas. There is a trend towards a more immunosuppressive phenotype in higher grade tumors with more regulatory T cells present in these tumor types. Both PD1 and PDL1 were present in only a small percentage of the tumor infiltrate. T cell receptor sequencing revealed up to 10% clonality of T cells in tumor infiltrates and no significant difference in clonality between low and high grade gliomas. We have shown the immune infiltrate of pediatric brain tumors does not appear to correlate with grade or survival for a small sample of patients. Further research and larger studies are needed to fully understand the interaction of pediatric brain tumors and the immune system.

Prospective feasibility and safety assessment of surgical biopsy for patients with newly diagnosed diffuse intrinsic pontine glioma

Background Diagnosis of diffuse intrinsic pontine glioma (DIPG) has relied on imaging studies, since the appearance is pathognomonic, and surgical risk was felt to be high and unlikely to affect therapy. The DIPG Biology and Treatment Study (DIPG-BATS) reported here incorporated a surgical biopsy at presentation and stratified subjects to receive FDA-approved agents chosen on the basis of specific biologic targets. Methods Subjects were eligible for the trial if the clinical features and imaging appearance of a newly diagnosed tumor were consistent with a DIPG. Surgical biopsies were performed after enrollment and prior to definitive treatment. All subjects were treated with conventional external beam radiotherapy with bevacizumab, and then stratified to receive bevacizumab with erlotinib or temozolomide, both agents, or neither agent, based on O6-methylguanine-DNA methyltransferase status and epidermal growth factor receptor expression. Whole-genome sequencing and RNA sequencing were performed but not used for treatment assignment. Results Fifty-three patients were enrolled at 23 institutions, and 50 underwent biopsy. The median age was 6.4 years, with 24 male and 29 female subjects. Surgical biopsies were performed with a specified technique and no deaths were attributed to the procedure. Two subjects experienced grade 3 toxicities during the procedure (apnea, n = 1; hypertension, n = 1). One subject experienced a neurologic deficit (left hemiparesis) that did not fully recover. Of the 50 tumors biopsied, 46 provided sufficient tissue to perform the study assays (92%, two-stage exact binomial 90% CI: 83%-97%). Conclusions Surgical biopsy of DIPGs is technically feasible, associated with acceptable risks, and can provide biologic data that can inform treatment decisions.