DNA was extracted from samples of primary brain tumor and xenografts and from patient-matched normal blood lymphocytes obtained from the Tissue Bank at the Preston Robert Tisch Brain Tumor Center at Duke University and collaborating centers, as described previously.17 (link) All analyzed brain tumors were subjected to consensus review by two neuropathologists. Table 1 lists the types of brain tumors we analyzed. The samples from glioblastomas included 138 primary tumors and 13 secondary tumors. Of the 138 primary tumors, 15 were from patients under the age of 21 years. Secondary glioblastomas were categorized as WHO grade IV on the basis of histologic criteria but had been categorized as WHO grade II or III at least 1 year earlier. Of the 151 tumors, 63 had been analyzed in our previous genomewide mutation analysis of glioblastomas. None of the lower-grade tumors were included in that analysis.16 (link)
In addition to brain tumors, we analyzed 35 lung cancers, 57 gastric cancers, 27 ovarian cancers, 96 breast cancers, 114 colorectal cancers, 95 pancreatic cancers, and 7 prostate cancers, along with 4 samples from patients with chronic myelogenous leukemia, 7 from patients with chronic lymphocytic leukemia, 7 from patients with acute lymphoblastic leukemia, and 45 from patients with acute myelogenous leukemia. All samples were obtained in accordance with the Health Insurance Portability and Accountability Act. Acquisition of tissue specimens was approved by the institutional review board at the Duke University Health System and at each of the participating institutions.
Exon 4 of the IDH1 gene was amplified with the use of a polymerase-chain-reaction (PCR) assay and sequenced in DNA from the tumor and lymphocytes from each patient, as described previously.16 (link) In all gliomas and medulloblastomas without an R132 IDH1 mutation, exon 4 of the IDH2 gene (which contains the IDH2 residue equivalent to R132 of IDH1) was sequenced and analyzed for somatic mutations. In addition, we evaluated all astrocytomas and oligodendrogliomas of WHO grade I to grade III, all secondary glioblastomas, and 96 primary glioblastomas without R132 IDH1 mutations or R172 IDH2 mutations for alterations in the remaining coding exons of IDH1 and IDH2. All coding exons of TP53 and PTEN were also sequenced in the panel of diffuse astrocytomas, oligodendrogliomas, anaplastic oligodendrogliomas, anaplastic astrocytomas, and glioblastomas. EGFR amplification and the CDKN2A-CDKN2B deletion were analyzed with the use of quantitative real-time PCR in the same tumors.18 (link) We evaluated samples of oligodendrogliomas and anaplastic oligodendrogliomas for loss of heterozygosity at 1p and 19q, as described previously.15 (link),19 (link)
In addition to brain tumors, we analyzed 35 lung cancers, 57 gastric cancers, 27 ovarian cancers, 96 breast cancers, 114 colorectal cancers, 95 pancreatic cancers, and 7 prostate cancers, along with 4 samples from patients with chronic myelogenous leukemia, 7 from patients with chronic lymphocytic leukemia, 7 from patients with acute lymphoblastic leukemia, and 45 from patients with acute myelogenous leukemia. All samples were obtained in accordance with the Health Insurance Portability and Accountability Act. Acquisition of tissue specimens was approved by the institutional review board at the Duke University Health System and at each of the participating institutions.
Exon 4 of the IDH1 gene was amplified with the use of a polymerase-chain-reaction (PCR) assay and sequenced in DNA from the tumor and lymphocytes from each patient, as described previously.16 (link) In all gliomas and medulloblastomas without an R132 IDH1 mutation, exon 4 of the IDH2 gene (which contains the IDH2 residue equivalent to R132 of IDH1) was sequenced and analyzed for somatic mutations. In addition, we evaluated all astrocytomas and oligodendrogliomas of WHO grade I to grade III, all secondary glioblastomas, and 96 primary glioblastomas without R132 IDH1 mutations or R172 IDH2 mutations for alterations in the remaining coding exons of IDH1 and IDH2. All coding exons of TP53 and PTEN were also sequenced in the panel of diffuse astrocytomas, oligodendrogliomas, anaplastic oligodendrogliomas, anaplastic astrocytomas, and glioblastomas. EGFR amplification and the CDKN2A-CDKN2B deletion were analyzed with the use of quantitative real-time PCR in the same tumors.18 (link) We evaluated samples of oligodendrogliomas and anaplastic oligodendrogliomas for loss of heterozygosity at 1p and 19q, as described previously.15 (link),19 (link)
