Shiyang Wang

Therapeutic and prognostic implications of BRAF V600E in pediatric low-grade gliomas

Purpose BRAF V600E is a potentially highly targetable mutation detected in a subset of pediatric low-grade gliomas (PLGGs). Its biologic and clinical effect within this diverse group of tumors remains unknown. Patients and Methods A combined clinical and genetic institutional study of patients with PLGGs with long-term follow-up was performed (N = 510). Clinical and treatment data of patients with BRAF V600E mutated PLGG (n = 99) were compared with a large international independent cohort of patients with BRAF V600E mutated-PLGG (n = 180). Results BRAF V600E mutation was detected in 69 of 405 patients (17%) with PLGG across a broad spectrum of histologies and sites, including midline locations, which are not often routinely biopsied in clinical practice. Patients with BRAF V600E PLGG exhibited poor outcomes after chemotherapy and radiation therapies that resulted in a 10-year progression-free survival of 27% (95% CI, 12.1% to 41.9%) and 60.2% (95% CI, 53.3% to 67.1%) for BRAF V600E and wild-type PLGG, respectively ( P < .001). Additional multivariable clinical and molecular stratification revealed that the extent of resection and CDKN2A deletion contributed independently to poor outcome in BRAF V600E PLGG. A similar independent role for CDKN2A and resection on outcome were observed in the independent cohort. Quantitative imaging analysis revealed progressive disease and a lack of response to conventional chemotherapy in most patients with BRAF V600E PLGG. Conclusion BRAF V600E PLGG constitutes a distinct entity with poor prognosis when treated with current adjuvant therapy.

Programmed death ligand 1 expression and tumor infiltrating lymphocytes in neurofibromatosis type 1 and 2 associated tumors

Immune checkpoint inhibitors targeting programmed cell death 1 (PD-1) or its ligand (PD–L1) have been shown to be effective in treating patients with a variety of cancers. Biomarker studies have found positive associations between clinical response rates and PD–L1 expression on tumor cells, as well as the presence of tumor infiltrating lymphocytes (TILs). It is currently unknown whether tumors associated with neurofibromatosis types 1 and 2 (NF1 and NF2) express PD–L1. We performed immunohistochemistry for PD–L1 (clones SP142 and E1L3N), CD3, CD20, CD8, and CD68 in NF1-related tumors (ten dermal and six plexiform neurofibromas) and NF2-related tumors (ten meningiomas and ten schwannomas) using archival formalin-fixed paraffin-embedded tissues. Expression of PD–L1 was considered positive in cases with > 5% membranous staining of tumor cells, in accordance with previously published biomarker studies. PD–L1 expression in tumor cells (using the SP142 and E1L3N clones, respectively) was assessed as positive in plexiform neurofibromas (6/6 and 5/6) dermal neurofibromas (8/10 and 6/10), schwannomas (7/10 and 10/10), and meningiomas (4/10 and 2/10). Sparse to moderate presence of CD68, CD3, or CD8 positive TILs was found in 36 (100%) of tumor specimens. Our findings indicate that adaptive resistance to cell-mediated immunity may play a major role in the tumor immune microenvironment of NF1 and NF2-associated tumors. Expression of PD–L1 on tumor cells and the presence of TILs suggest that these tumors might be responsive to immunotherapy with immune checkpoint inhibitors, which should be explored in clinical trials for NF patients.

Recurrent homozygous deletion of DROSHA and microduplication of PDE4DIP in pineoblastoma

Pineoblastoma is a rare and highly aggressive brain cancer of childhood, histologically belonging to the spectrum of primitive neuroectodermal tumors. Patients with germline mutations in DICER1, a ribonuclease involved in microRNA processing, have increased risk of pineoblastoma, but genetic drivers of sporadic pineoblastoma remain unknown. Here, we analyzed pediatric and adult pineoblastoma samples (n = 23) using a combination of genome-wide DNA methylation profiling and whole-exome sequencing or whole-genome sequencing. Pediatric and adult pineoblastomas showed distinct methylation profiles, the latter clustering with lower-grade pineal tumors and normal pineal gland. Recurrent variants were found in genes involved in PKA- and NF-κB signaling, as well as in chromatin remodeling genes. We identified recurrent homozygous deletions of DROSHA, acting upstream of DICER1 in microRNA processing, and a novel microduplication involving chromosomal region 1q21 containing PDE4DIP (myomegalin), comprising the ancient DUF1220 protein domain. Expresion of PDE4DIP and DUF1220 proteins was present exclusively in pineoblastoma with PDE4DIP gain.Pineoblastoma is a highly aggressive and rare childhood brain cancer, and the genetic drivers of sporadic pineoblastoma are unknown. Here, the authors genomically interrogated pediatric and adult pineoblastomas and found novel variants including recurrent homozygous deletions of DROSHA.

Pre-treatment lymphopenia and indication of tumor-induced systemic immunosuppression in medulloblastoma

The presence of tumor-induced systemic immune suppression, including lymphopenia, has been recognized in adult patients with glioblastoma for several decades, and pre-treatment neutrophil-to-lymphocyte count ratio (NLCR) is associated with inferior clinical outcome in patients with glioblastoma. Whether tumor-induced systemic immune suppression is also present in children with malignant brain tumors is not known. We performed a retrospective analysis of pretreatment neutrophil and lymphocyte counts in pediatric patients with medulloblastoma (MB) compared to a control group of children with posterior fossa pilocytic astrocytoma (PA). Compared to the control group, we observed statistically significantly lower absolute lymphocyte counts (ALCs) and higher NLCRs in the medulloblastoma group. Our findings suggest the presence of tumor-induced systemic immune suppression in MB patients already present at the time of diagnosis, with potential implications for the development of immune therapies in this population.

Abstract PR01: A DNA methylation-based classifier for accurate molecular diagnosis of bone sarcomas

Background: Bone sarcomas present a unique diagnostic challenge because of the considerable morphologic overlap between different entities. The choice of optimal treatment, however, is dependent upon accurate diagnosis. Genome-wide DNA methylation profiling has emerged as a new approach to aid in the diagnosis of brain tumors, with diagnostic accuracy exceeding standard histopathology. In this work we developed and validated a methylation-based classifier to differentiate between osteosarcoma, Ewing9s sarcoma, and synovial sarcoma. Methods: DNA methylation status of 482,421 CpG sites in 15 osteosarcoma, 10 Ewing9s sarcoma, and 11 synovial sarcoma samples were measured using the Illumina HumanMethylation450 array. From this training set of 36 sarcoma samples we developed a random forest classifier from the 400 most differentially methylated CpG sites (FDR q value Results: Methylation profiling revealed three distinct molecular clusters, each enriched with a single sarcoma subtype. Within the validation cohorts, all samples from TCGA were accurately classified as synovial sarcoma (10/10, sensitivity and specificity 100%). All but one sample from TARGET-OS were classified as osteosarcoma (85/86, sensitivity 98%, specificity 100%) and all but one sample from the Ewing9s sarcoma series was classified as Ewing9s (14/15, sensitivity 93%, specificity 100%). The single misclassified osteosarcoma sample was classified as a Ewing9s sarcoma, and later determined to be a misdiagnosed Ewing9s sarcoma based on RNA-seq data demonstrating high EWRS1 and ETV1 expression. An additional clinical sample that was misdiagnosed as a synovial sarcoma based on initial histopathology was accurately recognized as osteosarcoma by the methylation classifier. Pathway analysis with MSigDB (Broad Institute) identified targets of polycomb group proteins SUZ12 and EED, possessing the H3K27 trimethylated mark, as highly enriched within classifier genes Conclusions: Osteosarcoma, Ewing9s sarcoma, and synovial sarcoma have distinct epigenetic profiles. Our validated methylation-based classifier provides increased diagnostic accuracy, including in cases where standard histopathology is inconclusive. This abstract is also being presented as Poster A01. Citation Format: Shengyang Wu, Benjamin Cooper, Fang Bu, Christopher Bowman, Jonathan K. Killian, Shiyang Wang, Twana Jackson, Daniel Gorovets, Richard Gorlick, Kristen Thomas, Matthias Karajannis, Matija Snuderl. A DNA methylation-based classifier for accurate molecular diagnosis of bone sarcomas [abstract]. In: Proceedings of the AACR Conference on Advances in Sarcomas: From Basic Science to Clinical Translation; May 16-19, 2017; Philadelphia, PA. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(2_Suppl):Abstract nr PR01.

A DNA methylation-based classifier for accurate molecular diagnosis of bone sarcomas.

Purpose Pediatric sarcomas provide a unique diagnostic challenge. There is considerable morphologic overlap between entities, increasing the importance of molecular studies in the diagnosis, treatment, and identification of therapeutic targets. We developed and validated a genome-wide DNA methylation based classifier to differentiate between osteosarcoma, Ewings sarcoma, and synovial sarcoma. Materials and Methods DNA methylation status of 482,421 CpG sites in 10 Ewings sarcoma, 11 synovial sarcoma, and 15 osteosarcoma samples were determined using the Illumina Infinium HumanMethylation450 array. We developed a random forest classifier trained from the 400 most differentially methylated CpG sites within the training set of 36 sarcoma samples. This classifier was validated on data drawn from The Cancer Genome Atlas (TCGA) synovial sarcoma, TARGET Osteosarcoma, and a recently published series of Ewings sarcoma. Results Methylation profiling revealed three distinct patterns, each enriched with a single sarcoma subtype. Within the validation cohorts, all samples from TCGA were accurately classified as synovial sarcoma (10/10, sensitivity and specificity 100%), all but one sample from TARGET-OS were classified as osteosarcoma (85/86, sensitivity 98%, specificity 100%) and 14/15 Ewings sarcoma samples classified correctly (sensitivity 93%, specificity 100%). The single misclassified osteosarcoma sample demonstrated high EWSR1 and ETV1 expression on RNA-seq although no fusion was found on manual curation of the transcript sequence. Two additional clinical samples, that were difficult to classify by morphology and molecular methods, were classified as osteosarcoma when previously suspected to be a synovial sarcoma and Ewings sarcoma on initial diagnosis, respectively. Conclusion Osteosarcoma, synovial sarcoma, and Ewings sarcoma have distinct epigenetic profiles. Our validated methylation-based classifier can be used to provide diagnostic assistance when histological and standard techniques are inconclusive.