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Supplemental Table 2: List of SNPs analyzed.

Supplemental Table 3. Primer sequences.

Supplemental Table 1: Specimen Characteristics.

Identifying modifiers of glioma risk in patients with type I neurofibromatosis (NF1) could help support personalized tumor surveillance, advance understanding of gliomagenesis, and potentially identify novel therapeutic targets. Here, we report genetic polymorphisms in the human adenylate cyclase gene adenylate cyclase 8 (ADCY8) that correlate with glioma risk in NF1 in a sex-specific manner, elevating risk in females while reducing risk in males. This finding extends earlier evidence of a role for cAMP in gliomagenesis based on results in a genetically engineered mouse model (Nf1 GEM). Thus, sexually dimorphic cAMP signaling might render males and females differentially sensitive to variation in cAMP levels. Using male and female Nf1 GEM, we found significant sex differences exist in cAMP regulation and in the growth-promoting effects of cAMP suppression. Overall, our results establish a sex-specific role for cAMP regulation in human gliomagenesis, specifically identifying ADCY8 as a modifier of glioma risk in NF1. Cancer Res; 75(1); 16–21. ©2014 AACR.

Background: Cabozantinib, an oral FDA approved multi-receptor tyrosine kinase inhibitor, was tested in our preclinical mouse model of PN. After finding significant reduction of tumor number and size in cabozantinib treated versus control mice, we sought to translate these findings to a phase 2 human study. Here we report the activity of cabozantinib in adolescents and adults with NF1-associated PN. Methods: A multicenter, nonrandomized phase 2 trial (NCT02101736) of cabozantinib in subjects ≥16 years with NF1 and either progressive or clinically significant inoperable PN was performed by the NFCTC (NF-105). The primary study aim was volumetric response of the target PN determined by MRI read centrally. Cabozantinib was administered continuously for up to 24 cycles, each cycle was 28 days. The starting dose was 40 mg once daily with planned escalation to a target dose of 60 mg once daily after 2 cycles. Dose reductions for toxicity were allowed to 20 mg once daily. Partial response (PR) was defined as ≥20% reduction in tumor volume from baseline. Subjects were considered evaluable for response if they completed ≥1 cycle of therapy and had a follow-up MRI. Success was defined as ≥25% of subjects achieving and maintaining a PR after 12 cycles without significant toxicity. Investigation of the impact of cabozantinib on the PN kinome network was performed on murine samples. Results: Twenty-three subjects enrolled; 21 subjects (median age 22 years) were evaluable for toxicity (2 noted to be ineligible before receiving study drug) and 19 subjects (median age 23 years) were evaluable for response (1 subject withdrew during cycle 1 and 1 was found ineligible after starting study drug). Baseline median tumor size was 557 mL (range 57-2954 mL). Among the evaluable patients, 8 (42%) met criteria for PR by cycle 12. Median change in tumor volume was -15.2% (range +2.2% to -36.9%). No subject had PN progression on treatment; maximal tumor response was not achieved until at least 18 cycles in 6/8 responders. A significant portion of patients underwent dose reductions or discontinued cabozantinib due to low grade adverse events (AE) that impaired quality of life; however, 3 responders reduced to 20 mg maintained or improved their response at this dose. The most common AEs (any grade) in ≥10 patients included gastrointestinal toxicity, hypothyroidism, fatigue and palmar plantar erythrodysesthesia (PPE). Eleven grade 3 AEs occurred in 8 patients, mainly PPE (n=4) and hypertension (n=2); no grade 4 or 5 AEs occurred. Analysis of kinase activity in murine PN treated with cabozantinib showed significantly decreased activity of AXL, MERTK and MET, known cabozantinib targets, but also of DDR1 and DDR2. Conclusions: Cabozantinib demonstrates considerable clinical activity for PN with a radiographic response rate of 42%. Although there were few severe AEs, low grade toxicities impacted the willingness of many subjects to continue treatment. Quantitative kinome analysis revealed that inhibition of DDR1, DDR2, AXL, MERTK and MET might underpin the therapeutic responses seen in these patients. Lower doses of cabozantinib may be optimal for the NF1 population and still lead to therapeutic response. This trial is now enrolling a pediatric cohort of children aged 3 to 15 years. Supported by DOD Award W81XWH-12-1-0155 and Exelixis Citation Format: Chie-Schin Shih, Jaishri Blakeley, D. Wade Clapp, Amy E. Armstrong, Pam Wolters, Eva Dombi, Gary Cutter, Nicole J. Ullrich, Jeffrey Allen, Roger Packer, Stewart Goldman, David H. Gutmann, Scott Plotkin, Tena Rosser, Kent Robertson, Brigitte Widemann, Steven Rhodes, Steven Angus, Gary Johnson, Bruce Korf, Michael J. Fisher. Treatment of neurofibromatosis type 1 (NF1)-related plexiform neurofibromas (PN) with cabozantinib (XL184): A Neurofibromatosis Clinical Trials Consortium Phase II trial [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr CT233.

Introduction: Although molecular profiling is increasingly being applied to improve subgroup classification and to provide novel prognostic and predictive biomarkers, clinical neuropathology practice is largely based on morphology and immunohistochemistry. Current molecular methods play only a small role in determining the diagnosis itself. Methods: For molecular subclassification of tumors at NYU neuropathology and to improve diagnostic accuracy, we introduced genome-wide methylation profiling through Illumina Infinium HumanMethylation 450k array that can detect methylation marks from the DNA extracted from formalin-fixed paraffin embedded tissues. To this effort, an in-house pipeline was established in-house pipeline, which includes morphologic review, sample preparation, molecular profiling and bioinformatics analysis. We compared the methylation profiles to a reference cohort of 2150 cases from 77 tumor entities previously profiled and analyzed at German Cancer Research Center using a random forest algorithm and customized bioinformatics packages, which were shared between our institutions. Selected copy number variants (CNV) and mutations were confirmed by Fluorescence in situ Hybridization (FISH) or sequencing, and mutation specific immunohistochemistry, respectively. Results: We profiled 60 difficult in-house or consult adult and pediatric brain tumors where diagnosis, grade and/or molecular subtype were not conclusive by morphology, immunohistochemistry or standard molecular studies alone. There was 100% concordance with concurrently performed molecular tests such as 1p/19q, EGFR/BRAF CNV, MGMT promoter methylation or IDH1 status testing when these tests were performed for clinical care. Methylation profiling provided additional, relevant information in 30 of 60 (50%) cases, leading to a change of diagnosis in 9 (15%), clarification of the diagnosis in 7 (12%) cases, and further molecular subgroup refinement in 14 (23%) of cases, helping to direct further molecular testing and clinical management. Conclusion: The 450k methylation array platform represents a cost-efficient method to obtain molecular profiles of brain tumors to identify biologically relevant diagnostic subgroups, thereby improving diagnostic accuracy, and helping inform appropriate clinical management decisions. Citation Format: Kasthuri S. Kannan, Aristotelis Tsirigos, Jonathan Serrano, Lynn Ann Forrester, Arline Faustin, Cheddhi Thomas, David Capper, Volker Hovestadt, Stefan M. Pfister, David T. W Jones, Martin Sill, Daniel Schrimpf, Andreas von Deimling, Adriana Heguy, Sharon L. Gardner, Jeffrey Allen, Cyrus Hedvat, David Zagzag, Matija Snuderl, Matthias A. Karajannis. Advancing methylation profiling in neuropathology: Diagnosis and clinical management. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Integrating Clinical Genomics and Cancer Therapy; Jun 13-16, 2015; Salt Lake City, UT. Philadelphia (PA): AACR; Clin Cancer Res 2016;22(1_Suppl):Abstract nr 11.

INTRODUCTION: Pineoblastoma (PB) is one of the rarest and most aggressive brain tumors of childhood. PB is considered a “primitive neuroectodermal tumor” (PNET) based on histology, and commonly treated using treatment protocols developed for medulloblastoma; however the survival remains poor. A subset of PBs may occur in the setting of germline mutations involving DICER1 or RB1, but no next-generation sequencing studies have been published on PB to date, and the genetic drivers of sporadic PB remain unknown. METHODS: 21 tumor samples with a histological diagnosis of PB (including recurrent/metastatic samples) from 15 patients were included in this study. Matching germline DNA was available from 2 patients. We performed genome-wide methylation array profiling (Illumina Infinium 450k) on all samples, as well as whole-genome (for samples with matching germline DNA) or whole-exome sequence analysis. Fluorescence in situ hybridization (FISH) and digital droplet PCR (ddPCR) was performed to confirm select focal somatic gains. RESULTS: 14/18 samples from 9/13 patients analyzed by 450k profiling had a methylation signature similar to previously profiled PBs from a reference cohort. Samples from 4 patients were found to be more consistent with a diagnosis of embryonal tumor with multilayered rosettes (ETMR) - like tumor (non 19q amplified), papillary tumor of the pineal region, or pineal parenchymal tumor of intermediate differentiation, respectively. No mutations in DICER1 or RB1 were found. Homozygous deletions in DROSHA were found in tumors from 3 PB patients. In addition, we identified novel recurrent somatic gains involving chromosomal region 1q21 that were confirmed by FISH and ddPCR in 4/5 PB patients. CONCLUSION: Our studies revealed multiple candidate drivers of oncogenesis in PB. We identified novel homozygous deletions in DROSHA, a nuclease involved in microRNA processing. We also identified novel, highly recurrent somatic focal gains involving chromosomal region 1q21, which has been linked to brain growth, autism and schizophrenia, but not previously associated with cancer. Citation Format: Matija Snuderl, Kasthuri Kannan, Olga Aminova, Igor Dolgalev, Adriana Heguy, Arline Faustin, David Zagzag, Sharon L. Gardner, Jeffrey C. Allen, Jeffrey H. Wisoff, David Capper, Volker Hovestadt, Sama Ahsan, Charles Eberhart, Stefan M. Pfister, David T. w. Jones, Matthias A. Karajannis. Novel candidate oncogenic drivers in pineoblastoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr LB-172. doi:10.1158/1538-7445.AM2015-LB-172

Purpose: Intracranial germ cell tumors (GCTs) are rare brain tumors affecting primarily adolescents and young adults. They usually arise at the pineal or suprasellar region, where surgical intervention is very challenging. There are two major subtypes, germinoma and nongerminomatous GCT (NGGCT). The treatment of intracranial GCT (iGCT) is based on histologic classification and therefore correct classification is vital to the improvement of the outcome. However, due to frequent mixed histology and relatively low sensitivity and specificity of tumor markers, it is a challenging task to accurately subclassify iGCT based on limited biopsy. Recently, aberrant expression of microRNA (miRNA) in various cancers has been reported and thus miRNA holds great promise as a biomarker for cancer diagnosis and classification as well as for understanding tumor biology. We did genome-wide miRNA expression profiling of 45 cases of iGCT and studied the utility of miRNA as diagnostic markers and classifier in iGCT. Methods: Tumor samples were collected from Texas Children's Cancer Center and three other collaborating institutions. Expression of 367 miRNA was measured by real time qPCR TaqMan assay. Differentially expressed miRNAs were used to train various supervised multivariate classification algorithms. Leave-one-out cross validation was used to estimate the overall accuracy, sensitivity and specificity of the classifier. We used a reiterative process to reduce the number of miRNAs in the classifier without sacrificing its discriminating power. Result: Using 33 fresh frozen and 23 formalin-fixed paraffin-embedded (FFPE) samples (11 cases with matched samples), each set of samples showed distinct clusters of germinomas and NGGCTs by unsupervised clustering. We confirmed that good quantification of miRNA expression was possible by using FFPE samples, which are generally more challenging to use for mRNA expression profiling. The classifier based on 7 miRNAs profiled in FFPE samples showed high sensitivity (87.5%) and specificity (100%) in distinguishing germinomas from NGGCTs. The same 7-miRNA classifier identified in FFPE series was able to distinguish the subtypes in fresh frozen samples with 93.3% sensitivity and 87.5% specificity. Similarly, the classifier based on 5 miRNAs profiled in fresh frozen samples was able to classify the subtypes in FFPE samples accurately. Many highly expressed miRNAs in iGCT are identical to those found in embryonal stem cells (ESCs). It is interesting to note that the miRNAs that are differentially expressed between two subtypes of iGCTs are similar to those that show significant changes in expression when undifferentiated ESCs undergo induced differentiation in vitro. Conclusion: We demonstrated that miRNA expression in FFPE specimens could be used in clinical diagnosis and classification of iGCT. Our results also suggest that iGCT and ESCs show similar miRNA profiles and could have implications on the cell of origin of iGCTs. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1144. doi:10.1158/1538-7445.AM2011-1144