Mutation profiling of pleomorphic xanthoastrocytoma and giant cell glioblastoma arising in a PXA reveals BRAF V600E mutations.

<p>The sections on the left illustrate SNaPshot genotyping and the sections on the right depict Sanger sequencing of <i>BRAF</i> exon 15 for the same samples. The top panel shows genotyping data obtained with normal male genomic DNA (Promega, Madison, WI). The lower panels illustrate BRAF V600E (c.1799T>A) mutation detection (arrows) in tumor DNA derived from formalin-fixed paraffin-embedded specimens of representative examples of: PXA (BT21), anaplastic PXA (BT22) and gcGBM arising in a PXA (BT49). Assays: (1) <i>EGFR</i> 2235_49del R; (2) <i>NRAS</i> 38; (3) <i>BRAF</i> 1799; (4) <i>NRAS</i> 182; (5) <i>PIK3CA</i> 263; (6) <i>TP53</i> 742; (7) <i>CTNNB1</i> 95 and (8) <i>CTNNB1</i> 122.</p

Mutation analysis of GBM.

<p>SNaPshot clinical genotyping of 62 glioblastoma cases from MGH identified cancer gene mutations that confer a favorable prognosis to the patients (IDH1) or that activate pathways targeted by therapeutic agents under clinical development (BRAF, KRAS and PIK3CA).</p

Representative photomicrographs of tumors.

<p>A. H&E-stained section of a pleomorphic xanthoastrocytoma (PXA, BT06) demonstrating fascicular growth pattern and prominent intercellular reticulin deposition (B) corresponding to PXA (m). C. H&E-stained section of PXA (BT21) that demonstrates neither a fascicular growth pattern nor prominent intercellular reticulin deposition (D). E. H&E-stained section of a giant cell glioblastoma (gcGBM) arising from a PXA (BT49) with marked pleomorphism and giant cells and multifocal reticulin deposition (F).</p

Clinical Data and Summary of Results.

<p>PXA – pleomorphic xanthoastrocytoma; PXA (m) – pleomorphic xanthoastrocytoma with mesenchymal-like growth pattern; aPXA – anaplastic pleomorphic xanthoastrocytoma; NA – Not assessed.</p

Overview of proposed methods for FSM ULS processing of FFPE specimens.

<p>Summary of methods and timeline for aCGH using the FSM method. Following DNA extraction, the workflow and protocol for preparation of fresh or frozen samples is identical to FFPE workflow shown.</p

Size matching using FSM is a more critical determinant of array quality than other known variables.

<p>(A–H) Probe log₂ ratio (signal intensity test DNA/signal intensity reference DNA) data plotted for a single chromosome (chr.13 or chr.1) from eight Agilent 1 M arrays (green;log₂ratio<-0.3, black;-0.3≤log₂ratio≤0.3, red;log₂ratio>0.3). (A–C) Chromosome 13 plotted log₂ ratios are representative profiles of three Agilent 1 M arrays of a single FFPE GBM specimen (GBM1) processed with the FSM ULS protocol (A), standard ULS protocol (B), or FSM ULS protocol after altered proteinase K digestion during DNA extraction (C) (plotted log₂ ratio data for all chromosomes provided in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038881#pone.0038881.s002" target="_blank">Figure S2</a>). (D–H) Chromosome 1 plotted log₂ ratios are representative profiles of five Agilent 1 M arrays of a single FFPE GBM specimen (GBM2) processed using the FSM ULS protocol, with reduced DNA input in (E) and (F) (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038881#pone.0038881.s003" target="_blank">Figure S3</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038881#pone.0038881.s004" target="_blank">Figure S4</a> for detailed copy number analysis). Increased hybridization time (G) improved quality to a modest degree. Use of FFPE brain tissue as reference DNA (H) did not significantly improve results (dLRsd of 0.21 vs. 0.20 for standard reference).</p

Predicted hierarchy of known variables contributing to aCGH data quality.

<p>Predicted hierarchy of known variables contributing to aCGH data quality.</p

A Fragmentation Simulation Method (FSM) enables accurate prediction and precise control of labeled DNA fragment sizes.

<p>(A–F) Vertical axes indicate DNA bp. (A,D) Gel image of DNA from three FFPE specimens (A) or three frozen specimens (D) either intact, (i), after ULS labeling conditions only, (0), or ULS labeling conditions and 0.5, 1, 2, 4, 6, or eight minutes heat fragmentation (0.5, 1, 2, 4, 6, 8). (B,E) Utilizing mode fragment size of lanes in (A) or (D) as data points, FSM regression curves fit to data from each sample. Intersection with target size (dashed line) reveals FSM prediction for optimal time of heat fragmentation for each sample. (C,F) Agarose gel electrophoresis of samples in (A) or (D) after heat fragmentation for time predicted by FSM in (B) or (E) and ULS labeling conditions, shown adjacent to ImageJ gel analysis of same lanes. The mode fragment size of each smear, as measured with ImageJ, is indicated by arrows and solid horizontal lines.</p

Determination of optimal size among matched DNA fragment size distributions.

<p>(A,C,E,G) Agarose gel electrophoresis of reference gDNA (Promega) aliquots after various heat fragmentation times shown adjacent to ImageJ gel analysis of same lanes, molecular weight indicated in bp. Mode fragment size of each smear, as measured with ImageJ, indicated in blue. (B,D,F,H) Agilent 180 K array results of self-hybridizations using reference gDNA (left) and characterized by matching fragment size distributions (B;250/250, D;315/315, F;400/400, H;525/525). Log₂ ratios for signal intensities of differentially labeled aliquots (cy5/cy3) are plotted for probes corresponding to chromosome 1 (green;log₂ratio<−0.3, black;-0.3≤log₂ratio≤0.3, red;log₂ratio>0.3). Data quality was assessed by dLRsd. (I) Mean dLRsd of duplicate (n = 5) or triplicate (n = 2) size-matched self-hybridizations representing seven fragment size distributions plotted by mode fragment length (225, 250, 315, 400, 525, 625, and 680 bp). Error bars indicate SEM.</p

Application of FSM ULS method to FFPE samples creates equivalent results to those from fresh-frozen samples.

<p>(A) Plot showing dLRsd for 122 FFPE tumor specimens processed according to either standard ULS or FSM ULS protocols and analyzed on Agilent 1 M arrays. (B) Data quality (dLRsd) from (A) plotted by FFPE block age and method. Dashed lines indicate linear regression. Statistics indicate magnitude and significance of correlation between block age and aCGH data quality. (C) Quality (dLRsd) of Agilent 1 M aCGH data of 78 fresh-frozen tissue specimens or frozen tumorsphere cell cultures processed according to either standard ULS or FSM ULS protocols. (D) FFPE and Frozen FSM ULS subsets from (A) and (C) compared to 206 fresh-frozen GBM specimens analyzed on Agilent 244 k arrays from the glioblastoma TCGA study. Statistical significance was assessed by t test and ANOVA, (****;p<.0001, ns;p>0.05), and error bars indicate mean and standard deviation. Additional QC metrics data for all samples are provided in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038881#pone.0038881.s006" target="_blank">Table S2</a>.</p