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Blood, Vol. 114, Issue 3, 647-650, July 16, 2009
High frequency of PTEN, PI3K, and AKT abnormalities in T-cell acute lymphoblastic leukemia
Blood Gutierrez et al.
114: 647
Supplemental materials for: Gutierrez et al
Patient samples. T-ALL diagnostic specimens were collected with informed consent and Institutional Review Board approval from 49 children treated on Children’s Oncology Group 9404 (n=42) or Dana-Farber Cancer Institute 00–001 (n=7) clinical trials, which are similar therapeutic regimens based on an identical backbone including post-induction consolidation with asparaginase and anthracycline.1,2 Samples were purified to greater than 90% lymphoblasts by Ficoll-Hypaque centrifugation. Genomic DNA was extracted with the PureGene kit according to the manufacturer’s instructions (Gentra Systems, Minneapolis, MN). Array CGH. Microarray-based comparative genomic hybridization (array CGH) was performed on genomic DNA with Human Genome CGH 244A microarrays (Agilent Technologies, Santa Clara, CA), as previously described.3,4 Feature extraction data were obtained with Agilent G2567AA feature extraction software, normalized using a LOcally-WEighted regression Scatterplot Smoother (LOWESS) available in an R package developed by the Lynda Chin laboratory (http://genomic.dfci.harvard.edu/Tools/Agilent_1.0.2.tar.gz), and segmented with the BioConductor DNAcopy package (www.bioconductor.org/packages/2.2/bioc/html/DNAcopy.html). Note that samples 36 and 37 were excluded from analysis because CGH quality controls failed. Array CGH data are available on the GEO website under accession numbers GSE14959 (COG T-ALL cases, n=40) and GSE7615 (DFCI T-ALL cases, n=7). The CGH cutoff for PTEN deletion was defined as a segmented CGH log2 copy number ratio of less than −0.5 involving PTEN coding sequence. Fluorescence in situ hybridization. FISH was performed with a commercial probe mix containing a PTEN (10q23) probe labeled with SpectrumOrange and a chromosome 10 centromeric probe labeled with SpectrumGreen (Abbott Molecular, Abbott Park, IL). Slides and probes were co-denatured at 75°C, hybridized overnight at 37°C, washed, and counterstained with 4′-6-diamidino-2-phenylindole (DAPI). Images were obtained with an Axio Imager A1 fluorescence microscope (Carl Zeiss, Thornwood, NJ) using a 100X Alpha Apochromatic Plan oil immersion objective (Carl Zeiss, Thornwood, NJ), a JAI CV-M4+CL progressive scan camera (JAI Inc., San Jose, CA) and Genus acquisition software version 3.92 build 7 (Genetix USA, Boston, MA). Mutation detection. Sequencing was performed on the entire coding region of PTEN together with the selected exons of PI3K, AKT, RAS, PTPN11, NOTCH1 and FBXW7 detailed in the results section, using genomic DNA from 44 of the 47 primary T-ALL samples that were successfully analyzed by CGH. All sequencing was performed at Agencourt Bioscience Corporation, Beverly, MA. Statistical analysis. Event-free survival was analyzed by the Kaplan-Meier method. Survival curves were generated with GraphPad Prism version 4.01 (GraphPad Software, La Jolla, CA), and differences between groups were compared by the Gehan-Breslow-Wilcoxon test. REFERENCES 1. Winter SS, Jiang Z, Khawaja HM, et al. Identification of genomic classifiers that distinguish induction failure in T-lineage acute lymphoblastic leukemia: a report from the Children’s Oncology Group. Blood. 2007;110:1429–1438.
2. Clinicaltrials.gov identifier: NCT00165178. http://clinicaltrials.gov/ct2/show/NCT00165178?term=NCT00165178&rank=1.
3. O’Neil J, Tchinda J, Gutierrez A, et al. Alu elements mediate MYB gene tandem duplication in human T-ALL. J Exp Med. 2007;204:3059–3066.
4. Maser RS, Choudhury B, Campbell PJ, et al. Chromosomally unstable mouse tumours have genomic alterations similar to diverse human cancers. Nature. 2007;447:966–971.
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