Study subjects and sample preparations
NF1 patients who satisfied the National Institutes of Health diagnostic criteria (NIH Consensus Statement) and who had developed either benign or malignant tumors were recruited. Tumor specimens were obtained from patients who undergo surgical resection at the Chang Gung Memorial Hospital. All patients provided informed written consent for their tissues to be used in this study, which was approved by the Chang Gung Medical Foundation Institutional Review Board. The corresponding clinical data were collected and used in accordance with proved protocols from Chang Gung Medical Foundation Institutional Review Board. Patient-matched lymphocyte DNA were also included to determine the germline event(s) of these NF1-associated tumors. Genomic DNA from archived tumor samples were prepared by PUREGENE DNA purification kit fit from GENTRA according to the manufacturer’s instruction.
Targeted gene sequencing using the IT-PGM Sequencing system
A custom designed NGS panel including five common NF1-related genes: NF1 (NM_000267, 17q11.2), SPRED1 (NM_152594, 15q14), KRAS (NM_004985, 12p12.1), BRAF (NM_004333, 7q34), and TP53 (NM_000546, 17p13.1) was performed for the mutation analysis in this study. The total length is 32.3 kb, 296 amplicons and the coverage is 507x. Template preparation and emulsion PCR, and Ion Sphere Particles (ISP) enrichment were performed on an Ion One Touch System by using the Ion OneTouch™ 200 Template Kit Version 2.0 (Life Technologies) according to the manufacturer’s instructions. The raw PGM data were processed with Torrent Suite v3.4.2 (Life Technologies) to generate sequence reads filtered by the pipeline software quality controls. The Integrative Genomics Viewer16 (Broad Institute, Cambridge, Massachusetts) was then used to visualize variants against the reference genome and for the analysis of depth coverage, sequence quality, and variant identification. Variant frequencies >1% in the dbSNP-Asian database were further filtered.
PCR amplification and Sanger sequencing verification
Fragments with putative NF1 mutations or potential mutations were amplified by PCR and sequenced with Applied Biosystems 3730 Genetic Analyzer in DNA Sequencing Service of Chang Gung Memorial Hospital. A Standard Sanger sequencing was also performed exon by exon for SUZ12. PCR amplification were carried out under standard conditions with 30 PCR cycles and 55°-60°C annealing. PCR products will then be sequenced using the Big Dye Terminator cycle sequencing kit (Life Technologies) according to manufacturer’s cycling conditions. Sequence alignments and analysis were further performed using the Autoassembler computer program (Life Technologies).
Molecular inversion probe (MIP) DNA array analysis
The Affymetrix OncoScan Assay utilizing the Molecular Inversion Probe (MIP) technology was performed for larger copy number alterations and loss of heterozygosity (LOH). This OncoScan assay contains more than 300,000 copy number and single nucleotide polymorphism (SNP) oligonucleotide probes with a median probe spacing of 4,200 kilobases (kb), with much denser coverage within known cancer genes. Twenty-four available tumor DNA samples (80 ng genomic DNA) was performed following protocols provided by manufacturer (Affymetrix, Santa Clara, CA, USA). Briefly, samples were split to separate (A/T) and (G/C) channels. After circularization, MIPs were linearized, cleaved and then amplified by PCR. Amplicons were then cleaved into two fragments (44 bp) with HaeIII. DNA fragments were subsequently hybridized to OncoScan® arrays at 49 °C and 60 rpm for 16-18 h. The hybridized array were then washed, stained and scanned through GeneChip® scanner 3000 7G (Affymetrix, CA). Array fluorescence intensity data (CEL files), generated by Affymetrix® GeneChip® Command Console® (AGCC, Affymetrix) Software version 1.1 were processed to produce OSCHP files and QC metrics. Samples passing QC criteria (MAPD ≤ 0.3, ndSNPQC ≥ 26) were further analyzed through tumor Scan (TuScan) and BioDiscovery's SNPFASST2 algorithm using the Nexus Express for Oncoscan software 3.0 (Biodiscovery, Hawthorne, CA) and whole chromosome gains and losses and copy number aberrations (deletions and duplications) were determined.
Protein expression through immunohistochemistry in different NF1- associated tumors
Parafﬁn-embedded surgical resection specimens were retrospectively collected from the archives of the Tissue Bank at Department of Pathology, Chang Gung Memorial Hospital, for subsequent molecular analysis. Five micrometer sections mounted onto poly-L-lysine coated slides were deparaffinized and rehydrated in graded alcohols and distilled water. Endogenous peroxidase activity were quenched by incubation in 3% hydrogen peroxide in methanol for 20 min. Antigen retrieval were done by microwaving at high power for two cycles of 5 min each with a 10 min break between cycles in citrate buffer at pH 6.0. Slides were incubated with each primary antibodies at 4℃ overnight. A mouse monoclonal antibody NF1 (A-12) (Sc-74445; Santa Cruz Biotechnology, Santa Cruz, CA), targeting amino acids 1-300 of the NF1 protein, was used as NF1 primary antibody. The SUZ12-antibodies used in this study was mouse monoclonal antibody against clone SUZ220A (ab126577; Abcam, Cambridge, UK). Immunohistochemistry for H3K27me3 was performed using a rabbit polyclonal antibody directed against the trimethylated lysine 27 residue of the N-terminal portion of histone H3 (1:500 dilution; Cat.# 07-449; Millipore, Billerica, MA, USA). Following the incubation of primary antibodies, these slides were then incubated with horseradish peroxidase-avidin-biotin complex (Vectastain ABC Elite, Vector Laboratories). The enzyme-binding site were visualized by 3, 3-diaminobenzidine (DAB-kit, Vector Laboratories). Non-specific binding of secondary antibody were then blocked by incubating in 10% normal horse serum in PBS containing 1% BSA. Finally, the slides were counterstained with hematoxylin and dehydrated in graded alcohol; air dried, and mounted using Resin-based mounting medium under coverslips. The results of immunoreactivity of each antibody were evaluated by pathologist and scored according to the percentage of tumor cells with positive nuclear immunostaining and staining intensity. Each sample was scored as follows: －, negative; 1＋, 5% positive cells; 2＋, 6-50% positive cells; 3＋, >50% positive cells.
SPSS 13.0 software package was used for all statistical analyses. For evaluation of the difference in percentage of numbers of mutation, the Mann-Whitney U test was performed. A P value of <0.05 was considered to be statistical significance.