Comment[ArrayExpressAccession] E-GEOD-45843 MAGE-TAB Version 1.1 Public Release Date 2013-04-23 Investigation Title Genomic analysis of diffuse pediatric low-grade gliomas identifies recurrent, oncogenic MYBL1-truncating rearrangements Comment[Submitted Name] Genomic analysis of diffuse pediatric low-grade gliomas identifies recurrent, oncogenic MYBL1-truncating rearrangements Experiment Description Pediatric low-grade gliomas (PLGGs) are among the most common solid tumors in children but apart from BRAF mutations or duplications in specific subclasses, few genetic driver events are known. Diffuse PLGGs comprise a set of uncommon subtypes that exhibit invasive growth and are therefore especially challenging clinically. These tumors are particularly poorly understood. We performed high-resolution copy-number analysis of 44 diffuse PLGGs to identify recurrent alterations. Diffuse PLGGs exhibited fewer such alterations than adult low-grade gliomas, but we identified several significantly recurrent events. The most significant event, 8q13.1 gains, were observed in 28% of diffuse astrocytomas grade II (DA2s) and resulted in partial duplication of the transcription factor MYBL1 with truncation of its C-terminal negative-regulatory domain. A similar recurrent deletion-truncation breakpoint was identified in two angiocentric gliomas in the related gene MYB on 6q23.3. Whole-genome sequencing of a MYBL1-rearranged DA2 demonstrated MYBL1 tandem duplication, and few other events. Two novel, truncated MYBL1 transcripts identified in this tumor induced anchorage-independent growth when expressed in 3T3 cells and tumor formation in nude mice. Truncated transcripts were also expressed in two additional tumors with MYBL1 partial duplication. Our results define clinically relevant molecular subclasses of diffuse PLGGs and highlight a potential role for the MYB family in the biology of low-grade gliomas. IRB approval from all institutions was obtained, and all samples were from patients who provided informed consent or were studied with waiver of the requirement for informed consent by the appropriate IRB. Samples of various histologic subtypes were identified and collected at multiple institutions (Boston Children’s Hospital, Boston, MA, The University of Texas School of Medicine Southwestern, Dallas, TX, Children’s Cancer Hospital, Egypt, Cairo, Johns Hopkins University School of Medicine, Baltimore, MD, Children’s National Medical Center, Washington, DC, The Hospital for Sick Children, Toronto, Canada, Mayo Clinic, Rochester, MN). Central histopathologic review was performed by at least three board-certified neuropathologists using WHO criteria. DNA extraction from archival FFPE samples and Array CGH were performed as previously described (Craig, et al., 2012 PLoS One). GC-normalized copy-number data for the samples were then cleaned of known germline CNVs. Circular Binary Segmentation was used to segment the copy-number data, using parameters [alpha=0.001, undo.splits=sdundo, undo.SD=1.5, min.width=5]. Forty-four samples passed QC metrics (based on aCGH quality metrics for DNA integrity) for inclusion in the GISTIC analysis. Segmented data were analyzed with GISTIC 2.0 to determine statistically significant recurrent broad and focal CNAs. The following parameters were used: minimum segment size = 8, lesion amplitude threshold = 0.2, focal/broad cutoff = 0.9x chromosome arm length, q-value threshold 0.10, and gene confidence level 0.95. Term Source Name ArrayExpress EFO Term Source File http://www.ebi.ac.uk/arrayexpress/ http://www.ebi.ac.uk/efo/efo.owl Person Last Name Horowitz Ramkissoon Horowitz Craig Ramkissoon Rich Schumacher McKenna Lawrence Bergthold Brastianos Tabak Ducar vanHummelen MacConaill Pouissant-Young Cho Taha Mahmoud Bowers Margraf Tabori Hawkins Packer Hill Pomeroy Eberhart Dunn Goumnerova Getz Chan Santagata Hahn Stiles Ligon Kieran Beroukhim Ligon Person First Name Peleg Lori Peleg Justin Shakti Benjamin Steve Aaron Mike Guillaume Priscilla Barbara Matthew Paul Laura Tina Yoon-Jae Hala Madeha Daniel Linda Uri Cynthia Roger Dana Scott Charles Ian Liliana Gad Jennifer Sandro William Charles Azra Mark Rameen Keith Person Mid Initials Moshe A M M H E E K D E C J A L G F A C D H W L Person Email geo@ncbi.nlm.nih.gov Person Affiliation Dana-Farber Cancer Institute Person Address Cancer Biology, Dana-Farber Cancer Institute, 44 Binney St. SM1010, Boston, MA, USA Person Roles submitter Protocol Name P-GSE45843-1 P-GSE45843-5 P-GSE45843-6 P-GSE45843-2 P-GSE45843-3 P-GSE45843-4 P-GSE45843-7 Protocol Description Detailed in Craig, et al., 2012 PLoS One. Scanned images were processed using Agilent Feature Extraction v10.7 and FE Protocol CGH_107_Sep09. Quality control dLRsd statistics were recorded as reported in the QC Metrics file generated by the software. Copy number analysis was performed using the DNA Analytics module of Agilent Genomic Workbench 6.5. Log2 ratios were corrected for a periodic ‘‘wave’’ artifact that correlates with GC content using the software’s GC correction tool with a GC window size of 2 kb. The ADM-2 algorithm was used with a threshold of 6.0 to detect significantly aberrant genomic regions and detected regions were filtered for those spanning more than five probes (,10 kb) with an average absolute log2 ratio .0.3. ID_REF = VALUE = normalized log2 ratio (test/reference) Detailed in Craig, et al., 2012 PLoS One. Purified DNA extracts from FFPE tissues, frozen tissues, and frozen cells were heat fragmented as indicated by FSM analysis. Subsequently, ULS labeling (Agilent Technologies, Genomic DNA ULS Labeling Kit cat. no. 5190-0419, Santa Clara, CA) was performed according to the manufacturer’s suggested protocol. Briefly, 2 ug DNA from each sample was combined with 2 uL ULS-Cy5 Reagent (Genomic DNA ULS Labeling Kit) and 2 uL 10X Labeling Solution (Genomic DNA ULS Labeling Kit) prior to 30 min at 85C and >10 min at 4C in a PCR thermocycler. An equal mass of either male or female reference DNA was heat-fragmented according to FSM predictions and then labeled with the ULS-Cy3 Reagent (Genomic DNA ULS Labeling Kit). Unincorporated dye was removed using Genomic DNA Purification Modules (Agilent Technologies, cat. no. 5190-0418). Detailed in Craig, et al., 2012 PLoS One. The entire volumes of the Cy5-labeled sample DNA and the Cy3-labeled reference DNA were combined together with 37.8 uL H2O, 50 uL Cot-1 DNA (Invitrogen, cat. no. 15279-011, Carlsbad, CA), 5.2 uL 100X Blocking Agent (Agilent Technologies, Oligo aCGH Hybridization Kit cat. no. 5188-5220), and 260 uL 2X Hi-RPM Hybridization Buffer (Agilent Technologies, cat. no. 5190-0403) before denaturation (3 min at 95C) and pre-hybridization (30 min at 37C). 130 uL Agilent-CGHblock (Agilent Technologies, cat. no. 5190-0421) was added to each hybridization solution before 490 uL of the combined solution was applied to a gasket slide (Agilent Technologies, cat. no. G2534-60003). A 1x1 M SurePrint G3 Human CGH Microarray (Agilent Technologies, cat. no. G4447A) was paired with each gasket slide in a SureHyb Enabled Hybridization Chamber (Agilent Technologies, cat. no. G2534A) and the differentially labeled DNA samples were hybridized (65C) to the microarray for 40–72 hrs in a hybridization oven (Agilent Technologies, cat. no. G2545A). During hybridization the slides were rotated at 19 rpm. NA NA Detailed in Craig, et al., 2012 PLoS One. Briefly, 1 mm cores (two to five cores total) or 20 um sections (three to five sections total) were taken from regions estimated to contain greater than 50% tumor cells based on previous pilot studies showing accurate detection of single copy gains and losses in samples with .40% tumor nuclei by pathologist estimate of H&E slides. Cores or sections were placed in sterile nuclease-free microcentrifuge tubes and paraffin was removed by treating the tissue in (1.2 ml) xylene. Samples were rinsed twice with 1.2 ml 100% ethanol and allowed to dry at room temperature before the addition of 0.9 ml 1 M NaSCN and overnight incubation at 37C. After 12–24 hrs, samples were rinsed twice in 0.9 ml 1X PBS. 0.34 ml of Buffer ATL (Qiagen, QIAamp DNA FFPE Tissue Kit cat. no. 56404, Valencia, CA) and 40 ml of Proteinase K (20 mg/ mL) (Qiagen, cat. no. 19131) were added and samples were incubated in a thermomixer (Eppendorf, cat. no. 022670000, Hamburg, Germany) set at 56–58C and 450 rpm. An additional 40 ul Proteinase K was added every 8–12 hrs for a period of 48–72 hrs. Samples were allowed to cool to room temperature before the addition of 10–20 ul RNase A (100 mg/mL) (Qiagen, cat. no. 19101) and a 5–10 minute incubation at room temperature. After adding 400 ul of Buffer AL (Qiagen QIAamp DNA FFPE Tissue Kit), samples were placed in thermomixer at 60C for 10 minutes. 440 ul of 100% ethanol was added and each sample was split between two QIAamp MinElute Columns (Qiagen QIAamp DNA FFPE Tissue Kit). Following successive washes with 500 ul Buffer AW1 (Qiagen QIAamp DNA FFPE Tissue Kit) and 500 ul 80% ethanol, DNA was eluted in 50–100 ul H2O. Detailed in Craig, et al., 2012 PLoS One. Microarrays and gaskets were disassembled at room temperature in Wash Buffer 1 (Agilent Technologies, cat. no. 5188-5221) and quickly moved to a second dish containing Wash Buffer 1 and a stir bar rotating at speed sufficient for gentle agitation of the liquid’s surface. After 5–30 minutes, slides were moved to a dish containing Wash Buffer 2 (Agilent Technologies, cat. no. 5188-5222) and a stir bar and agitated at 37C for 1 minute. Slides were then washed in anhydrous acetonitrile (Sigma- Aldrich, cat. no. 271004, St. Louis, MO) for 10–15 sec before being removed and placed in a slide holder (Agilent Technologies, cat. no. G2505-60525) with an Ozone-Barrier Slide Cover (Agilent Technologies, cat. no. G2505-60550). Microarrays were scanned immediately with a DNA Microarray Scanner (Agilent Technol- ogies, cat. no. G2505C) at 3 um resolution. Protocol Type normalization data transformation protocol labelling protocol hybridization protocol sample treatment protocol growth protocol nucleic acid extraction protocol array scanning protocol Experimental Factor Name TISSUE TYPE Experimental Factor Type tissue type Comment[SecondaryAccession] GSE45843 Comment[GEOReleaseDate] 2013-04-23 Comment[ArrayExpressSubmissionDate] 2013-04-08 Comment[GEOLastUpdateDate] 2013-04-23 Comment[AEExperimentType] comparative genomic hybridization by array SDRF File E-GEOD-45843.sdrf.txt