healthmedicinet

Study materials and clinical data

Study cases were retrieved from the surgical pathology files of the Department of
Pathology and Laboratory Medicine, Taipei Veterans General Hospital (VGH-TPE), Taiwan.
The Parent/legal guardian of patients in this study provided informed consent, and
all procedures were approved by the Institutional Review Board of VGH-TPE (VGHIRB
No.:2011-11-007GA 2011-11-008GA). Fresh tumor tissues that were removed during surgery
were snap-frozen and stored in liquid nitrogen until DNA and RNA extraction. The overall
survival time was calculated as the time from surgery until death or the time from
surgery until the last follow-up appointment for the patients who survived. A Mann–Whitney
test was used to compare age differences among the different groups of patients. The
differences in survival times were assessed with the log-rank test.

Histopathologic diagnosis of AT/RT and MB

A diagnosis of AT/RT was based on the morphologic features of the tumor and the results
of IHC, as described in our previous reports 7], 14]. With regard to the morphologic features, rhabdoid cells, which were either large,
pale, bland cells or the classical type similar to those observed in malignant rhabdoid
tumors of the kidney, were essential for diagnosis 8], 15]. Other features, such as cells that are similar to primitive neuroectodermal cells,
an epithelial component and a mesenchymal component, could also be observed. An IHC
diagnostic panel included the following markers: epithelial membrane antigen [EMA;
monoclonal, dilution 1:40, Dako, Glostrup, Denmark, Histostain SP Broad Spectrum (HRP),
Zymed Lab., Carlsbad, USA (Histostain); antigen retrieval using a microwave, three
cycles for 5 min each (M)], vimentin (VIM; monoclonal, 1:600, Dako, Histostain, M),
smooth muscle actin (SMA, HHF-35; monoclonal, 1:75, Dako, Carpinteria, CA, USA, Histostain,
M), and glial fibrillary acidic protein (GFAP; monoclonal, 1:300, Dako, Histostain,
M) 5], 8], 14]. The rhabdoid cells were immunoreactive for two or more of the above-listed antibodies.
IHC for INI1 (anti-BAF47; monoclonal, 1:40, BD Transduction Laboratories, San Diego,
CA, USA, Histostain, M) and SMARCA4 (anti-BRG1; monoclonal, 1:100, Abcam, Cambridge,
U.K., Histostain, M) was included in all cases in this study. With regard to a diagnosis
of MB, besides the presence of the known morphology of this tumor type, the features
of AT/RT as listed above were absent. Immunostaining for synaptophysin (SYN; monoclonal,
1:50, Novocastra, Newcastle upon Tyne, U.K., Histostain, M) was included to confirm
the diagnosis and to distinguish it from AT/RT; IHC for SYN was also performed in
all AT/RT cases. Positive and negative controls were included with each batch of sections
to confirm the consistency of the analysis in all of the stains that were performed
in this study. As for the INI1 staining, positive control consisted of endothelial
cells within the tumor. Negative controls consisted of staining without applying the
primary antibody and staining of a known INI1 negative AT/RT.

Direct sequencing, reverse transcription-PCR (RT-PCR) and Quantitative real-time reverse
transcription-PCR (qRT-PCR)

Genomic DNAs and total RNA were isolated from fresh-frozen tumor samples by the DNeasy
Blood Tissue Kit and RNAeasy (Qiagen) according to the manufacturer’s instruction
(Qiagen, GmbH, Germany), respectively. Genomic DNAs were used to peform PCR using
specific INI1 gene primers and then sequenced by direct sequencing. For RT-PCR and
qRT-PCR, 1 ?g of total RNA was used to perform reverse transcription (RT) using the
RevertAidâ„¢ Reverse transcriptase kit (Cat. K1622; Fermentas, Glen Burnie, Maryland,
USA) as directed by the manufacturer. For INI1 RT-PCR, a paired-primer encompassing
exon 5 and 6 was used. For INI1, the forward primer was 5?-AACAGGAACCGCATGGGCCG-3?,
and the reverse primer was 5?-GCCCGTGTTCCGGATGGCAA-3? (amplicon size: 579 bps). For
GAPDH, the forward primer was 5?-CAAGGTCATCCATGACAACTTTG-3?, and the reverse primer
was 5?-GTCCACCACCCTGTTGCTGTAG-3? (amplicon size, 496 bps). Quantitative real-time
PCR reactions were performed using Maximaâ„¢ SYBR Green qPCR Master Mix (Cat. K0222;
Fermentas, Glen Burnie, Maryland, USA), and the specific products were detected and
analyzed using the StepOneâ„¢ sequence detector (Applied Biosystems, USA). The expression
level of each gene was normalized to GAPDH expression. For GAPDH, the forward primer
was 5?-CCAGCCGAGCCACATCGCTC-3? and the reverse primer was 5?-ATGAGCCCCAGCCTTCTCCAT-3?.
For SOX4, the forward primer was 5?- TCGCTGTACAAGGCGCGGAC-3? and the reverse primer
was 5?-TTCTCCGCCAGGTGCTTGCC-3?. For ERBB2, the forward primer was 5?- AGTACCTGGGTCTGGACGTG-3?
and the reverse primer was 5?-CTGGGAACTCAAGCAGGAAG-3?. For OLIG2, the forward primer
was 5?-CAGAAGCGCTGATGGTCATA-3? and the reverse primer was 5?-TCGGCAGTTTTGGGTTATTC-3?.

Array CGH (aCGH) analysis

As described in our previous study 14], the samples were mixed with control DNA samples from healthy donors before they
were subjected to the analysis. A Human Genome CGH Microarray Kit 244A (Agilent Technologies,
USA) with 99,000 probes and an average probe spatial resolution of 15.0 kb was used.
aCGH was performed according to the protocol suggested by Agilent. Data analysis was
performed using CGH Analytics 3.4 (Agilent Technologies) using the default parameters.
Briefly, chromosomal abbreviations were calculated using the ADM2 statistic algorithm
with a moving average window of 1 Mb; additionally, the default thresholds of ADM2
recommended by Agilent were used to make an amplification or deletion call.

Gene expression microarray (GEM) and computational analyses

Array data on adult neural stem cells and embryonic stem cells were obtained in our
previous study 16] and from the Gene Expression Omnibus (GEO; http://www.ncbi.nlm.nih.gov/geo/) dataset GSE9940. An mRNA expression array analysis was performed as previously described
16], 17]. Briefly, an Affymetrixâ„¢ HG-U133 Plus 2.0 whole genome array was used. RMA log expression
units were calculated from the Affymetrix GeneChip array data with the ‘affy’ package
of the Bioconductor (http://www.bioconductor.org/) suite software for the R statistical programming language (http://www.r-project.org/). The default RMA settings were used to background correct, normalize and summarize
all expression values. Significant differences between sample groups were identified
by the ‘limma’ package 16]. Briefly, a t-statistic was calculated as normal for each gene, and a p-value was
then calculated with a modified permutation test 16]. To control for the multiple testing errors, a false discovery rate (FDR) algorithm
was then applied to these p-values to calculate a set of q-values: thresholds of the
expected proportion of false positives or false rejections of the null hypothesis.
Heat maps were then created by dChip software (http://www.dchip.org/). Classical multidimensional scaling (MDS) was performed with the standard function
of the R program to provide a visual impression of how the various sample groups are
related. Gene annotation was performed by the ArrayFusion web tool (http://microarray.ym.edu.tw/tools/arrayfusion/) 18]. Principal component analysis (PCA) was performed with Partek Genomics Suite software
(http://www.partek.com) to provide a visual impression of how the various sample groups are related. All
array data have been submitted to the NCBI Gene Expression Omnibus (GEO) database,
and the accession number is GSE65132 (Additional file 2).

MicroRNA microarray analysis

The Agilent Human miRNA Microarray Kit V2 (Agilent, Foster City, CA, USA) containing
probes for 723 human microRNAs from the Sanger database v10.1 was used. GeneSpring
GX 9 software (Agilent, USA) was used for value extraction. A 2-tailed Student’s t-test was then used for the calculation of the p value for each miRNA probe.