iplex genotyping IDH1 and IDH2 assays utilized the following primer sets (forward and reverse primers along with extension primers).

Transcription

1 Supplementary Materials Supplementary Methods iplex genotyping IDH1 and IDH2 assays utilized the following primer sets (forward and reverse primers along with extension primers). IDH1 R132H and R132L Forward: 5 -ACGTTGGATGCAACATGACTTACTTGATCCCC-3 Reverse: 5 -ACGTTGGATGAAAAATATCCCCCGGCTTGTG-3 Extension: 5 -GTAAAACCTATCATCATAGGTC-3 IDH1 R132C, R132G, and R132S Forward: 5 -ACGTTGGATGAAAAATATCCCCCGGCTTGTG-3 Reverse: 5 -ACGTTGGATGCAACATGACTTACTTGATCCCC-3 Extension: 5 -ATCCCCATAAGCATGAC-3 IDH2 R172G Forward: 5 -ACGTTGGATGAAAAACATCCCACGCCTAGTC-3 Reverse: 5 -ACGTTGGATGTCAGTGGATCCCCTCTCCACC-3 Extension: 5 -GGTCGCCATGGGCGTGCC-3 IDH2 R172K and R172M Forward: 5 -ACGTTGGATGTCAGTGGATCCCCTCTCCACC-3 Reverse: 5 -ACGTTGGATGAAAAACATCCCACGCCTAGTC-3 Extension: 5 -GCCCATCACCATTGGCA-3 Multiplexed PCR was performed in 5 µl containing 0.1 U of Kapa 2G Fast Taq polymerase (Kapa Biosystems, Capetown, South Africa), 10 ng of genomic DNA, 2.5 pmol of each PCR primer and 2.5 mmol of dntp. An amplification

2 round to generate mutational hot spot regions was carried out using the following thermocycling conditions are as follows: 1 cycle of 95 C for 5 min; 3 cycles of 95 C for 30 sec, 64 C for 30 sec, 72 C for 60 sec; 3 cycles of 95 C for 30 sec, 62 C for 30 sec, 72 C for 60 sec; 3 cycles of 95 C for 30 sec, 60 C for 30 sec, 72 C for 60 sec; 37 cycles of 95 C for 30 sec, 58 C for 30 sec, 72 C for 60 sec, and one final cycle of 70 C for 5 min. One ml of a 1/10 dilution of final product was used as a template for a second round of amplification using the PCR conditions described above. Unincorporated dntps were deactivated using 0.3 U of shrimp alkaline phosphatase at 37º C for 40 minutes, followed by heat inactivation at 85º C for 5 minutes. Single-base primer extension was carried out using 5.4 pmol of each extension primer, 50 mmol of the appropriate dntp/ddntp combination and 0.5 units of Thermosequenase DNA polymerase (iplex enzyme). Extension reactions were cycled using a 200-short-cycle program that uses two cycling loops as follows: the sample is denatured at 94º C, annealing takes place at 52º C for 5 seconds and extension at 80º C for 5 seconds. The annealing and extension cycle is repeated four more times for a total of five cycles and then looped back to a 94º C denaturing step for 5 seconds and then enters the 5 cycle annealing and extension loop again. The five annealing and extension steps with the single denaturing step are repeated 40 times. The 40 cycles of the 5 cycle annealing and extension steps equate to a total of 200 cycles. A final extension is done at 72º C for three minutes and then the sample is cooled to 4º C. After the addition of a cation exchange resin to remove residual salt from the reactions, 10 nl of the

3 purified primer extension reaction were loaded onto a matrix pad (3- hydroxypicoloinic acid) of a SpectroCHIP (Sequenom). SpectroCHIPs were analyzed using a Bruker Biflex III matrix-assisted laser desorption/ionization time of flight (MALDI-TOF) mass spectrometer (SpectroREADER, Sequenom). All results were manually inspected, using the Mass ARRAY TyperAnalyzer v4.0 software. Automated mutation calling for TP53 sequencing Bi-directional reads and mapping tables (to link read names to sample identifiers, gene names, read direction, and amplicon) were subjected to a QC filter which excludes reads that have an average phred score of < 10 for bases Passing reads were assembled against the reference sequences for each gene, containing all coding and UTR exons including 5Kb upstream and downstream of the gene, using command line Consed 16.0 (PMID: ). Assemblies were passed on to Polyphred 6.02b (PMID: ), which generated a list of putative candidate mutations, and to Polyscan 3.0 (PMID: ) which generated a second list of putative mutations. The lists were merged together into a combined report, and the putative mutation calls were normalized to + genomic coordinates and annotated using the Genomic Mutation Consequence Calculator (PMID: ). The resulting list of annotated putative mutations was loaded into a Postgres database along with select assembly details for each mutation call (assembly position, coverage, and methods supporting mutation call). To reduce the number of false positives generated by the mutation detection software packages, only point mutations which are supported by at least one bi-directional

4 read pair and at least one sample mutation called by Polyphred were considered, and only the putative mutations which are annotated as having non-synonymous coding effects, occur within 11 bp of an exon boundary, or have a conservation score > ( were included in the final candidate list. Indels called by any method were manually reviewed and included in the candidate list if found to hit an exon. All traces for mutation calls were manually reviewed.

5 Supplementary Table Legends Table S1: Gene list of refined signature designating expression subclasses in lower-grade diffuse astrocytic gliomas from MSKCC sample set. ROC scores are shown. Table S2: Modified MSKCC sample set gene list used to analyze REMBRANDT expression data. ROC scores are shown. Table S3: Gene list of unified expression signature developed from subclass assignments in both MSKCC and REMBRANDT array data. Summed ROC scores are shown. Table S4: Ingenuity analysis results for canonical pathways using NB subclass Table S5: Ingenuity analysis results for functional annotations using NB subclass Table S6: Ingenuity analysis results for canonical pathways using EPL subclass Table S7: Ingenuity analysis results for functional annotations using EPL subclass

6 Table S8: Ingenuity analysis results for canonical pathways using PG subclass Table S9: Ingenuity analysis results for functional annotations using PG subclass Table S10: Demographic and molecular features of diffuse astrocytic gliomas (MSKCC sample set) stratified by primary/recurrent tumor status. P values are shown. Mt: mutant, Wt: wild-type, IHC: immunohistochemistry, unkn: unknown, FISH: fluorescence in-situ hybridization, NB: neuroblastic, EPL: early progenitorlike, PG: pre-gbm. Table S11: Histopathological features of diffuse astrocytic gliomas (MSKCC sample set) stratified by molecular subclass, WHO grade, and primary/recurrent tumor status. P values are shown. Pro: prominent (characterizing >50% of tumor), Pre: present, Ab: absent, NB: neuroblastic, EPL: early progenitor-like, PG: pre-gbm. Table S12: Statistically significant copy number alterations as determined by the GISTIC algorithm for REMBRANDT diffuse astrocytic gliomas of NB subclass. Table S13: Statistically significant copy number alterations as determined by the GISTIC algorithm for REMBRANDT diffuse astrocytic gliomas of EPL subclass.

7 Table S14: Statistically significant copy number alterations as determined by the GISTIC algorithm for REMBRANDT diffuse astrocytic gliomas of PG subclass. Supplementary Figure Legends FIG. S1: Kaplan-Meier curves showing overall survival for recurrent MSKCC diffuse astrocytic gliomas stratified by WHO grade and prior treatment modality (Surg: surgery only; Surg + Adj: surgery plus adjuvant radiation therapy and/or chemotherapy). Sample sizes are in parentheses. P=0.083 for WHO II tumors and P=0.363 for WHO III tumors. FIG. S2: Representative immunohistochemical stains and scoring. All photomicrographs were taken at 400X magnification. A, p53 immunopositivity required nuclear staining in >30% of tumor cells. B, PDGFRA immunopositivity required detectable staining, and was frequently seen in only a subset of tumor cells, particularly in IDH mt tumors. C, Strong p-pras40 immunostaining required robust signal in the majority of tumor cells. D, IDH R132H immunopositivity required detectable staining. FIG. S3: Consensus k-means clustering demonstrated 3 robust subclasses of lower-grade diffuse astrocytic glioma. Cluster stability for filtered sets of 200, 250, 300, and 350 genes was maximal at k=3 as measured by the Δ(Gini) (A-D).

8 FIG. S4: Positive silhouette width for initial clusters of diffuse astrocytic glioma determined core subclass members, which were then used for downstream SAM and ROC analysis. FIG. S5: Kaplan-Meier curves showing overall survival for REMBRANDT diffuse astrocytic gliomas stratified by WHO grade. Sample sizes are in parentheses. P value shown.