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Cancer Discov. 2014 Aug;4(8):956-71. doi: 10.1158/2159-8290.CD-13-0879. Epub 2014 Jun 3.

EGFR variant heterogeneity in glioblastoma resolved through single-nucleus sequencing.

Author information

1
Broad Institute of Harvard and MIT, Cambridge, Massachusetts. Department of Medical Oncology, Dana-Farber Cancer Institute, Boston, Massachusetts.
2
Broad Institute of Harvard and MIT, Cambridge, Massachusetts.
3
Department of Medical Oncology, Dana-Farber Cancer Institute, Boston, Massachusetts.
4
Broad Institute of Harvard and MIT, Cambridge, Massachusetts. Department of Chemical Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts. The David H. Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, Massachusetts.
5
Center for Molecular Oncologic Pathology, Dana-Farber Cancer Institute, Boston, Massachusetts.
6
Center for Molecular Oncologic Pathology, Dana-Farber Cancer Institute, Boston, Massachusetts. Department of Pathology, Brigham and Women's Hospital, Boston, Massachusetts. Department of Pathology, Boston Children's Hospital, Boston, Massachusetts. Department of Pathology, Harvard Medical School, Boston, Massachusetts.
7
Broad Institute of Harvard and MIT, Cambridge, Massachusetts. Department of Medical Oncology, Dana-Farber Cancer Institute, Boston, Massachusetts. Department of Pathology, Harvard Medical School, Boston, Massachusetts. Center for Cancer Genome Discovery, Dana-Farber Cancer Institute, Boston, Massachusetts. Keith_Ligon@dfci.harvard.edu Matthew_Meyerson@dfci.harvard.edu.
8
Department of Medical Oncology, Dana-Farber Cancer Institute, Boston, Massachusetts. Center for Molecular Oncologic Pathology, Dana-Farber Cancer Institute, Boston, Massachusetts. Department of Pathology, Brigham and Women's Hospital, Boston, Massachusetts. Department of Pathology, Boston Children's Hospital, Boston, Massachusetts. Department of Pathology, Harvard Medical School, Boston, Massachusetts. Keith_Ligon@dfci.harvard.edu Matthew_Meyerson@dfci.harvard.edu.

Abstract

Glioblastomas (GBM) with EGFR amplification represent approximately 50% of newly diagnosed cases, and recent studies have revealed frequent coexistence of multiple EGFR aberrations within the same tumor, which has implications for mutation cooperation and treatment resistance. However, bulk tumor sequencing studies cannot resolve the patterns of how the multiple EGFR aberrations coexist with other mutations within single tumor cells. Here, we applied a population-based single-cell whole-genome sequencing methodology to characterize genomic heterogeneity in EGFR-amplified glioblastomas. Our analysis effectively identified clonal events, including a novel translocation of a super enhancer to the TERT promoter, as well as subclonal LOH and multiple EGFR mutational variants within tumors. Correlating the EGFR mutations onto the cellular hierarchy revealed that EGFR truncation variants (EGFRvII and EGFR carboxyl-terminal deletions) identified in the bulk tumor segregate into nonoverlapping subclonal populations. In vitro and in vivo functional studies show that EGFRvII is oncogenic and sensitive to EGFR inhibitors currently in clinical trials. Thus, the association between diverse activating mutations in EGFR and other subclonal mutations within a single tumor supports an intrinsic mechanism for proliferative and clonal diversification with broad implications in resistance to treatment.

SIGNIFICANCE:

We developed a novel single-cell sequencing methodology capable of identifying unique, nonoverlapping subclonal alterations from archived frozen clinical specimens. Using GBM as an example, we validated our method to successfully define tumor cell subpopulations containing distinct genetic and treatment resistance profiles and potentially mutually cooperative combinations of alterations in EGFR and other genes.

PMID:
24893890
PMCID:
PMC4125473
DOI:
10.1158/2159-8290.CD-13-0879
[Indexed for MEDLINE]
Free PMC Article

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