Prospective, high-throughput molecular profiling of human gliomas

Abstract

Gliomas consist of multiple histologic and molecular subtypes with different clinical phenotypes and responsiveness to treatment. However, enrollment criteria for clinical trials still largely do not take into account these underlying molecular differences. We have incorporated a high-throughput tumor genotyping program based on the ABI SNaPshot platform as well as other molecular diagnostic tests into the standard evaluation of glioma patients in order to assess whether prospective molecular profiling would allow rational patient selection onto clinical trials. From 218 gliomas we prospectively collected SNaPshot genotyping data on 68 mutated loci from 15 key cancer genes along with data from clinical assays for gene amplification (EGFR, PDGFRA, MET), 1p/19q co-deletion and MGMT promoter methylation. SNaPshot mutations and focal gene amplifications were detected in 38.5 and 47.1 % of glioblastomas, respectively. Genetic alterations in EGFR, IDH1 and PIK3CA closely matched frequencies reported in recent studies. In addition, we identified events that are rare in gliomas although are known driver mutations in other cancer types, such as mutations of AKT1, BRAF and KRAS. Patients with genetic alterations that activate signaling pathways were enrolled onto genetically selective clinical trials for malignant glioma as well as for other solid cancers. High-throughput molecular profiling incorporated into the routine clinical evaluation of glioma patients may enable the rational selection of patients for targeted therapy clinical trials and thereby improve the likelihood that such trials succeed.

Fig 1

Representative examples of SNaPshot tumor genotyping. The top panels show genotyping data obtained for normal male genomic DNA (Promega, Madison, WI) and the bottom panels show data derived from patient GBM tumors. Mutant and wild-type alleles are distinguished based on the slightly different positions and on the distinct colors of their corresponding peaks. Arrows indicate mutant alleles. a Identification of the IDH1 R132H (cDNA nucleotide change 395C>A) mutation in one GBM specimen. Assays: (1) IDH1 R132; (2) EGFR E746_A750; (3) EGFR L858; (4) CTNNB1 S45; (5) PIK3CA E542; (6) NRAS G12; (7) EGFR L861 and (8) AKT1 E17. b Identification of the PIK3CA H1047R (3140A>G) mutation in another GBM tumor. Assays: (1) PIK3CA H1047; (2) CTNNB1 G34; (3) BRAF V600; (4) NRAS G13; (5) PIK3CA Q546; (6) APC R1450 and (7) APC R1114

Fig 2

Detection of gene amplification by fluorescence in situ hybridization (FISH). Tumor cells show EGFR amplification in a (orange signals) and MET amplification in b (pink signals). The CEP7 chromosome (centromere control) is represented by aqua signals (white arrowheads). Nuclei are stained with 4′,6-diamidino-2-phenylindole