Clinical implementation of integrated whole-genome copy number and mutation profiling for glioblastoma

Abstract

Background: Multidimensional genotyping of formalin-fixed paraffin-embedded (FFPE) samples has the potential to improve diagnostics and clinical trials for brain tumors, but prospective use in the clinical setting is not yet routine. We report our experience with implementing a multiplexed copy number and mutation-testing program in a diagnostic laboratory certified by the Clinical Laboratory Improvement Amendments.

Methods: We collected and analyzed clinical testing results from whole-genome array comparative genomic hybridization (OncoCopy) of 420 brain tumors, including 148 glioblastomas. Mass spectrometry-based mutation genotyping (OncoMap, 471 mutations) was performed on 86 glioblastomas.

Results: OncoCopy was successful in 99% of samples for which sufficient DNA was obtained (n = 415). All clinically relevant loci for glioblastomas were detected, including amplifications (EGFR, PDGFRA, MET) and deletions (EGFRvIII, PTEN, 1p/19q). Glioblastoma patients ≤40 years old had distinct profiles compared with patients >40 years. OncoMap testing reliably identified mutations in IDH1, TP53, and PTEN. Seventy-seven glioblastoma patients enrolled on trials, of whom 51% participated in targeted therapeutic trials where multiplex data informed eligibility or outcomes. Data integration identified patients with complete tumor suppressor inactivation, albeit rarely (5% of patients) due to lack of whole-gene coverage in OncoMap.

Conclusions: Combined use of multiplexed copy number and mutation detection from FFPE samples in the clinical setting can efficiently replace singleton tests for clinical diagnosis and prognosis in most settings. Our results support incorporation of these assays into clinical trials as integral biomarkers and their potential to impact interpretation of results. Limited tumor suppressor variant capture by targeted genotyping highlights the need for whole-gene sequencing in glioblastoma.

Keywords: array CGH; clinical trials; genomics; genotyping; glioblastoma.

Fig. 1.

(A) Summary of brain tumor samples submitted for OncoCopy from 2012–2013. (B) Demographics of GBM patients in DF/BWCC cohort compared with TCGA. (C) GBM-associated focal genomic events identified in DF/BWCC adult GBM by OncoCopy compared with TCGA. (D) Summary of GBM-associated oncogenes and tumor suppressor genes detected by OncoMap compared with TCGA.

Fig. 2.

FISH validates genomically distinct tumor subclones. Representative images of 5 tumors (GBM02, 04, 05, 07, and 08) assessed by FISH for MYCN, EGFR, MET, MYC, and PDGFRA confirm the presence of tumor subpopulations that were identified as low-level gains by aCGH. (B) FISH analysis of low-level gains identified by aCGH in 8 GBM samples with tumor subpopulations.

Fig. 3.

Copy number aberrations across 148 GBM cases by aCGH. (A) Heat map demonstrating amplifications (red) and deletions (blue) among the DF/BWCC GBM cohort. (B) Focal GISTIC peaks with significance of q-value <0.1 and associated genes from 148 GBM cases.

Fig. 4.

Array CGH reveals distinct genomic profiles between GBM patients ≤40 years of age and those >40 years. (A) Heat map of amplifications (red) and deletions (blue) demonstrates enrichment for chromosome 7 amplifications, CDKN2A/B deletions on chromosome 9, and chromosome 10 deletions in GBM patients >40 years of age. (B) Recurrent arm-level events in GBM patients ≤40 years of age compared with patients >40 years. (C) GISTIC 2.0 peaks in GBM patients ≤40 years of age compared with patients >40 years.

Fig. 5.

GISTIC 2.0 analysis and comparision of DF/BWCC aCGH data with TCGA GBM SNP array data. (A) Significance (x-axis) of DF/BWCC deletions (dark blue) and amplifications (lavender) transposed onto TCGA data (light blue and red, respectively) across the genome (y-axis). (B) SCNA peaks with significantly different incidence between adult (>40 y) DF/BWCC patients and TCGA cohort, as defined by P-value <.05 (2-tailed Fisher's exact test with Bonferroni correction).

Fig. 6.

Integrative OncoPrint of OncoCopy and OncoMap data for 37 GBM patients. The circles indicate patients with both copy number alterations and a mutation in PTEN or RB1.