Scott Ryall

Genomic analysis of diffuse intrinsic pontine gliomas identifies three molecular subgroups and recurrent activating ACVR1 mutations

Diffuse intrinsic pontine glioma (DIPG) is a fatal brain cancer that arises in the brainstem of children, with no effective treatment and near 100% fatality. The failure of most therapies can be attributed to the delicate location of these tumors and to the selection of therapies on the basis of assumptions that DIPGs are molecularly similar to adult disease. Recent studies have unraveled the unique genetic makeup of this brain cancer, with nearly 80% found to harbor a p.Lys27Met histone H3.3 or p.Lys27Met histone H3.1 alteration. However, DIPGs are still thought of as one disease, with limited understanding of the genetic drivers of these tumors. To understand what drives DIPGs, we integrated whole-genome sequencing with methylation, expression and copy number profiling, discovering that DIPGs comprise three molecularly distinct subgroups (H3-K27M, silent and MYCN) and uncovering a new recurrent activating mutation affecting the activin receptor gene ACVR1 in 20% of DIPGs. Mutations in ACVR1 were constitutively activating, leading to SMAD phosphorylation and increased expression of the downstream activin signaling targets ID1 and ID2. Our results highlight distinct molecular subgroups and novel therapeutic targets for this incurable pediatric cancer.

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.

A comprehensive review of paediatric low-grade diffuse glioma: pathology, molecular genetics and treatment

Gliomas are the most common central nervous system neoplasms affecting children and can be both high- and low-grade. Paediatric low-grade glioma may be either World Health Organization grade I or grade II. Despite being classified as grade II diffuse astrocytoma, these neoplasms arising in children are distinct clinically and molecularly from their adult counterparts. They do not tend to progress to higher grade lesions and only rarely harbour an IDH mutation. Here, we review the clinical, histologic and molecular features of paediatric grade II diffuse glioma, highlighting their diagnostic criteria, prevalence across brain locations, their most common molecular features and how to test for them, and lastly the current status of therapeutic options available for their treatment.

H3 K27M mutations are extremely rare in posterior fossa group A ependymoma

BackgroundMutations in the tail of histone H3 (K27M) are frequently found in pediatric midline high-grade glioma’s but have rarely been reported in other malignancies. Recently, recurrent somatic nucleotide variants in histone H3 (H3 K27M) have been reported in group A posterior fossa ependymoma (EPN_PFA), an entity previously described to have no recurrent mutations. However, the true incidence of H3 K27M mutations in EPN_PFA is unknown.MethodsIn order to discern the frequency of K27M mutations in histone H3 in EPN_PFA, we analyzed 151 EPN_PFA previously profiled with genome-wide methylation arrays using a validated droplet digital PCR assay.ResultsWe identified only 1 case out of 151 EPN_PFA harboring the K27M mutation indicating that histone mutations are extremely rare in EPN_PFA. Morphologically, this single mutated case is clearly consistent with an ependymoma, and the presence of the K27M mutation was confirmed using immunohistochemistry.DiscussionK27M mutations are extremely rare in EPN_PFA. Routine evaluation of K27M mutations in EPN_PFA is of limited utility, and is unlikely to have any bearing on prognosis and/or future risk stratification.

Multiplex Detection of Pediatric Low-Grade Glioma Signature Fusion Transcripts and Duplications Using the NanoString nCounter System

Previous studies identified recurrent fusion and duplication events in pediatric low-grade glioma (pLGG). In addition to their role in diagnosis, the presence of these events aid in dictating therapy and predicting patient survival. Clinically, BRAF alterations are most commonly identified using fluorescent in situ hybridization (FISH). However, this method is costly, labor-intensive and does not identify nonBRAF events. Here, we evaluated the NanoString nCounter gene expression system for detecting 32 of the most commonly reported fusion/duplication events in pLGG. The assay was validated on 90 pLGG samples using FISH as the gold standard and showed sensitivity and specificity of 97% and 98%, respectively. We next profiled formalin-fixed paraffin-embedded preserved biopsy specimens from 429 pLGG cases. 171 (40%) of the cases within our cohort tested positive for a fusion or duplication event contained within our panel. These events, in order of prevalence, were KIAA1549-BRAF 16;9 (89/171, 52.0%), KIAA1549-BRAF 15;9 (42/171, 24.6%), KIAA1549-BRAF 16;11 (14/171, 8.2%), FGFR1-TACC1 17;7 (13/171, 7.6%), MYBL1 duplication (5/171, 2.9%), KIAA1549-BRAF 18;10 (4/171, 2.3%), KIAA1549-BRAF 15;11 (2/171, 1.2%), FAM131B-BRAF 2;9 (1/171, 0.6%), and RNF130-BRAF 3;9 (1/171, 0.6%). This work introduces NanoString as a viable clinical replacement for the detection of fusion and duplication events in pLGG.

Abstract B12: H3-K27M is a negative prognostic marker in high- and low-grade pediatric thalamic glioma

Pediatric gliomas are the most commonly diagnosed brain cancer in children, accounting for approximately 50% of all cases. Work by our group and others has revealed recurrent histone mutations in diffuse intrinsic pontine glioma (DIPG), astrocytomas that arise in the brainstem of children and boast a dismal median survival of less than one year. In ~70% of DIPG, a mutation in histone variant H3.3 or H3.1 at position 27 leads to substitution of a lysine by a methionine (K27M). When correlated to clinical data, patients harbouring H3-K27M mutations showed a worse prognosis in comparison to patients wild type for H3 (H3-WT), regardless of their histologic grade. Investigation into H3-K27M prevalence in pediatric gliomas revealed that they were not exclusively observed in the brainstem, but rather, in additional midline structures such as the thalamus. However, the clinical relevance of H3-K27M in other midline tumours has not yet been investigated. We hypothesized that H3-K27M would represent a negative prognostic marker in pediatric thalamic gliomas. We searched the Hospital for Sick Children9s tumour archives and obtained 86 radiologically confirmed cases of pre-treatment thalamic glioma. The cohort contained 46 male and 40 female patients with a combined median age of diagnosis and survival of 9.04 and 3.88 years, respectively. Of the 86 samples analyzed, 53 were histologically reviewed as low grade malignancies while 33 appeared high grade. Median survival for patients with high grade thalamic gliomas was 1.02 years vs 8.71 years for low grade thalamic glioma patients. Six (11.3%) low grade and 17 (51.5%) high grade gliomas tested positive for the H3-K27M mutation. Patients were clinically annotated for treatment, extent of resection, age of diagnosis and outcome (median follow-up of 10.3 years) and were used to determine the prognostic implications of H3-K27M. Kaplan-Meier survival analysis revealed significantly worse overall survival of thalamic glioma patients harbouring the H3-K27M mutation versus wild type samples (log rank p These findings provide the first evidence that H3-K27M is a negative prognostic indicator for pediatric thalamic glioma. Further, the survival difference between H3-K27M mutant low grade and high grade astrocytoma suggests differing biology and a role for “second hits” in dictating the aggressiveness of these tumours. Importantly, it supports prognostic subgrouping based on mutation status and histologic grade. Citation Format: Scott Ryall, Pawel Buczkowicz, Anthony Arnoldo, Rahul Krishnatry, Matthew Mistry, Joshua Rubin, Paul Steinbok, Uri Tabori, Cynthia Hawkins. H3-K27M is a negative prognostic marker in high- and low-grade pediatric thalamic glioma. [abstract]. In: Proceedings of the AACR Special Conference on Chromatin and Epigenetics in Cancer; Sep 24-27, 2015; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2016;76(2 Suppl):Abstract nr B12.

Detecting Stem Cell Marker Expression Using the NanoString nCounter System

The use of embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) has become commonplace in the study of neuronal development, physiology, disease modelling, and therapy development. Due to the transient nature of working with these cells, it is important to regularly confirm the cell status as a naive stem cell versus a more defined neural progenitor cell (NPC). Classically, this has been done using a panel of specific antibodies to test for the expression of transcription factors known to be observed in ESCs, but not NPCs. However, this method is both time consuming and expensive. Here, we describe the use of the NanoString nCounter system for determining the levels of expression of key transcription factors that will effectively aid in determining the state of your stem cell cultures.

Sarcoma Subgrouping by Detection of Fusion Transcripts Using NanoString nCounter Technology

Background NanoString technology is an innovative barcode-based system that requires less tissue than traditional techniques and can test for multiple fusion transcripts in a single reaction. The objective of this study was to determine the utility of NanoString technology in the detection of sarcoma-specific fusion transcripts in pediatric sarcomas. Design Probe pairs for the most common pediatric sarcoma fusion transcripts were designed for the assay. The NanoString assay was used to test 22 specific fusion transcripts in 45 sarcoma samples that had exhibited one of these fusion genes previously by reverse transcription polymerase chain reaction (RT-PCR). A mixture of frozen (n = 18), formalin-fixed, paraffin-embedded (FFPE) tissue (n = 23), and rapid extract template (n = 4) were used for testing. Results Each of the 22 transcripts tested was detected in at least one of the 45 tumor samples. The results of the NanoString assay were 100% concordant with the previous RT-PCR results for the tumor samples, and the technique was successful using both FFPE and rapid extract method. Conclusion Multiplexed interrogation for sarcoma-specific fusion transcripts using NanoString technology is a reliable approach for molecular diagnosis of pediatric sarcomas and works well with FFPE tissues. Future work will involve validating additional sarcoma fusion transcripts as well as determining the optimal workflow for diagnostic purposes.