Supplementary Data
1. Supplementary Methods
1.1 RNA Extraction, DNA Extraction and cDNA synthesis
Total RNA and DNA were isolated from frozen tumor samples. Tumor samples
were pulverized under liquid nitrogen, and total RNA and DNA were extracted using
Trizol reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s
recommendations. Both were dissolved in RNAse-free water. A260 and A280 were
used to quantify and determine the purity of the total RNA. One microgram of RNA
extracted from each sample was synthesized into double-stranded cDNA using the
SuperScript III Reverse Transcriptase Kit (Invitrogen, Carlsbad, CA, USA), according to
the manufacturer´s instructions.
1.2 RT-PCR assay
The KIAA1549-BRAF fusion variants and BRAF mutations were amplified by PCR
(polymerase chain reaction), using primers whose the sequences are listed at Table 1
based on a previously published protocol [7,28]. Primers were designed to comprise all
variants of fusion between exon 8 of BRAF and exon 16 of KIAA1549. Primer specificity
was confirmed by BLAST. PCR were performed in 50μL reaction using 2μL of cDNA and
5,0U of Platinum DNA Polymerase (Invitrogen, Carlsbad, CA, USA) by incubation at
94°C for 2 minutes, followed by 36 amplification cycles of 94°C for 30 seconds, 61°C for
30 seconds, and 72°C for 90 seconds, and a final extension step of 72°C for 2 minutes.
The quality of cDNA was validated using another PCR reaction with housekeeper gene
GAPDH. This reaction took place in 25μL of mixture reaction using 1μL of cDNA and
5,0U of Platinum DNA Polymerase (Invitrogen, Carlsbad, CA, USA) by incubation at
94°C for 2 minutes, followed by 36 amplification cycles of 94°C for 30 seconds, 61°C for
30 seconds, then 72°C for 90 seconds, and a final extension at 72°C for 5 minutes.
In each assay a sample without nucleic acid was included to verify the absence
of contamination. PCR products were analyzed by electrophoresis with 2.0% agarose
gels and stained with Gel Red (Biotium, Hayward, CA, USA).
1.3 PCR
Exon 4 genome DNA isolated of each sample was submitted to PCR
amplification for exon 4 of both IDH1 and IDH2 genes, using the Platinum DNA
Polymerase (5,0U) (Invitrogen, Carlsbad, CA, USA) according to manufacturer’s
instruction. Primers were designed with the assistance of Primer Express Software
(Applied Biosystems, Foster City, CA, USA), and their sequences are listed in Table 1.
For the IDH1 amplification, DNA was subjected to 39 cycles consisting of 30 seconds of
denaturation at 94°C, 40 seconds annealing at 54°C, and 40 seconds extension at 72°C.
For the IDH2 amplification, DNA was subjected to 39 cycles consisting of 30 seconds of
denaturation at 94°C, 35 seconds annealing at 54°C, and 40 seconds extension at 72°C.
The PCR amplicons were visualized in 2.0% agarose gel.
1.4 Sequencing KIAA1549-BRAF fusion gene, BRAFV600E and exon 4 of IDH1 and
IDH2 genes
The PCR products from IDH1, IDH2, KIAA1549-BRAF and BRAF (exon 15) were
sequenced to identify mutations. We used ExoSAP-IT (Affymetrix, Cleveland, OH, USA),
BigDye V3.1 (Applied Biosystem, Carlsbad, CA, USA) and BigDye XTerminator (Applied
Biosystem, Carlsbad, CA, USA), according to the manufacturer’s instructions, and
sequenced in a 3500 Genetic Analyzer (Applied Biosystem-Hitachi, Carlsbad, CA, USA).
1.5 Statistical Analysis
Data analysis was performed using GraphPad Prism version 5 (San Diego,
California, USA). Clinical variables tested in the presence of the fusion gene and gene
mutations were: gender, age at diagnosis, tumor grade, primary site, and occurrence
of relapse. Categorical data (gender, histological subtype, presence of fusion gene,
primary site, presence of mutation gene, occurrence of relapse) were studied using
Fisher exact tests. Overall survival (OS) curves were generated by applying the KaplanMeier method. OS was defined as the time from diagnosis until the date of either the
most recent follow-up or death. We used Kaplan-Meier method to assess OS in all
patients, with 95% CI (Confidence Interval). Log-rank and Gehan-Breslow-Wilcoxon
Tests were used to confirm the overall survival analyses. Statistical significance was
taken as p< 0.05.
1.6 Multiple amino acid sequence alignment
Amino acid sequence alignment of IDH1 from five species was obtained from
NCBI database (http://www. ncbi.nlm.nih.gov/protein/) as follows: Homo sapiens (GI:
49168486), Bos taurus (GI: 74354744), Mus musculus (GI: 57242927), Rattus
norvegicus (GI: 13928690) and Canis lupus (GI: 89573987). These sequences were
compared using a computer-based multiple sequence alignment program: CLC
Sequence Viewer 6.0 (CLC bio A/S, Katrinebjerg, Denmark).
2. Supplementary Figures
Figure 1: A) IDH1 gene mutation at R132H (c.395G>A). B) IDH1 gene mutation at R109K
(c.326G>A) C) IDH2 gene mutation at R172S (c.516G>C) and D) IDH2 gene mutation at
L143L (c.429G>C)
A
B
C
D)
Figure 2: Schematic representation of the IDH1 gene. Legend: Alignment of gene IDH1
sequence in 5 different species, showing that the mutated residues in our sample
(c.326G> A) are highly conserved