Intratumoral heterogeneity of receptor tyrosine kinases EGFR and PDGFRA amplification in glioblastoma defines subpopulations with distinct growth factor response Nicholas J. Szerlipa, Alicia Pedrazab, Debyani Chakravartyb, Mohammad Azimc, Jeremy McGuirec, Yuqiang Fangd, Tatsuya Ozawae, Eric C. Hollande,f,g,h, Jason T. Hused,h, Suresh Jhanward, Margaret A. Levershac, Tom Mikkelseni, and Cameron W. Brennanb,f,h,1 aDepartment of Neurosurgery, Wayne State University Medical School, Detroit, MI 48201; bHuman Oncology and Pathogenesis Program, cMolecular Cytogenetic Core Laboratory, dDepartment of Pathology, eCancer Biology and Genetics Program, fDepartment of Neurosurgery, gDepartment of Surgery, and hBrain Tumor Center, Memorial Sloan-Kettering Cancer Center, New York, NY 10065; and iDepartments of Neurology and Neurosurgery, Henry Ford Health System, Detroit, MI 48202 Edited by Webster K. Cavenee, Ludwig Institute, University of California at San Diego, La Jolla, CA, and approved December 29, 2011 (received for review August 29, 2011) Glioblastoma (GBM) is distinguished by a high degree of intra- demonstrated in GBM and has been shown to mediate resistance tumoral heterogeneity, which extends to the pattern of expression to single-RTK inhibition through “RTK switching” in cell lines and amplification of receptor tyrosine kinases (RTKs). Although (11). Although such RTK coactivation has been measured at the most GBMs harbor RTK amplifications, clinical trials of small-mole- protein level, its significance in maintaining tumor cell sub- cule inhibitors targeting individual RTKs have been disappointing to populations has not been established. date. Activation of multiple RTKs within individual GBMs provides We have previously reported prominent PDGFR activation by a theoretical mechanism of resistance; however, the spectrum of platelet-derived growth factor beta (PDGFB) ligand in GBMs EGFR MET fi functional RTK dependence among tumor cell subpopulations in with or ampli cation (12). We hypothesized that PDGFRA fi actual tumors is unknown. We investigated the pattern of hetero- this could lead to cases wherein is ampli ed along with EGFR MET geneity of RTK amplification and functional RTK dependence in either or in the same tumor, signifying that both RTKs contribute to positive growth advantage. Amplifications of MEDICAL SCIENCES GBM tumor cell subpopulations. Analysis of The Cancer Genome EGFR PDGFRA Atlas GBM dataset identified 34 of 463 cases showing independent and are often heterogeneously distributed in focal amplification of two or more RTKs, most commonly platelet- GBM, and retention of the amplicons is thought to depend on α positive selection pressure (13, 14). Finding cases of GBM with derived growth factor receptor (PDGFRA) and epidermal growth fi factor receptor (EGFR). Dual-color fluorescence in situ hybridization focal ampli cation of two or more RTKs affords an opportunity was performed on eight samples with EGFR and PDGFRA amplifica- to identify by FISH whether this positive selection favors am- plification of both receptors in the same cells or in different tion, revealing distinct tumor cell subpopulations amplified for only populations and, if the populations are distinct, whether they are one RTK; in all cases these predominated over cells amplified for fi regionally segregated in the tumor. both. Cell lines derived from coampli ed tumors exhibited geno- In this study we establish the prevalence of RTK coamplification type selection under RTK-targeted ligand stimulation or pharmaco- from analysis of data from The Cancer Genome Atlas (TCGA) logic inhibition in vitro. Simultaneous inhibition of both EGFR and pilot project, examine the anatomic/cellular relationship of EGFR PDGFR was necessary for abrogation of PI3 kinase pathway activity and PDGFRA amplifications in select cases by FISH, and in- in the mixed population. DNA sequencing of isolated subpopula- vestigate the phenomenon of in vitro selection of EGFR-and tions establishes a common clonal origin consistent with late or PDGFRA-amplified genotypes from genetically heterogeneous cell ongoing divergence of RTK genotype. This phenomenon is espe- lines derived from coamplified tumors. cially common among tumors with PDGFRA amplification: overall, 43% of PDGFRA-amplified GBM were found to have amplification of Results EGFR MET or the hepatocyte growth factor receptor gene ( ) as well. Common Focal RTK Amplifications in GBM Are Not Mutually Exclusive and Define Subpopulations. From the TCGA dataset, array com- glioma | glioblastoma genetics | mosaicism | amplicon parative genomic hybridization (aCGH) profiles of 463 GBMs were examined for focal copy number aberration (CNA) spanning lioblastoma (GBM) is the most common primary malignant EGFR, PDGFRA,orMET. CNA focality was determined by Gbrain tumor in adults and is characterized by histologic intra- a previously described algorithm, genome topography scan (GTS, tumoral heterogeneity, invasive growth patterns, and overall poor available as R package from http://cbio.mskcc.org/brennan; response to treatment with conventional radiation and chemo- SI Methods) (15). Amplifications or gains were scored for RTKs therapy (1, 2). The majority of primary GBMs harbor amplification commonly altered in GBM: EGFR, MET, and the region of and/or mutation of a receptor tyrosine kinase (RTK): either epi- chr4q12 spanning three neighboring RTKs PDGFRA, KIT, and dermal growth factor receptor (EGFR;40–50%), platelet-derived KDR (“PDGFRA-region”). GTS focality scores above 0.02, growth factor receptor α (PDGFRA; ≈15%), or the hepatocyte growth factor receptor (MET, ≈5%) (3, 4). The high prevalence of EGFR and PDGFRA alterations seen in GBM has nominated these Author contributions: N.J.S., A.P., D.C., E.C.H., and C.W.B. designed research; N.J.S., A.P., RTKs as priority candidates for therapeutic targeting. However, D.C., M.A., J.M., Y.F., T.O., J.T.H., M.A.L., and C.W.B. performed research; T.O., S.J., M.A.L., clinical trials of small-molecule inhibitors targeting EGFR and T.M., and C.W.B. contributed new reagents/analytic tools; N.J.S., A.P., D.C., E.C.H., J.T.H., PDGFR used as single therapy have shown little response in un- S.J., and C.W.B. analyzed data; and N.J.S. and C.W.B. wrote the paper. selected patients, and amplification status of the receptors has not The authors declare no conflict of interest. yetbeenfoundtobepredictive(5–10). This article is a PNAS Direct Submission. One hypothesis for the therapeutic failure of targeting a single Freely available online through the PNAS open access option. RTK in GBM even when the gene is amplified or mutated is that 1To whom correspondence should be addressed. E-mail: [email protected]. other RTKs may be concurrently activated in the same tumor. In This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. fact, concurrent phosphorylation of multiple RTKs has been 1073/pnas.1114033109/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1114033109 PNAS Early Edition | 1of6 Downloaded by guest on September 29, 2021 implying CNA shared with 50 or fewer neighboring genes, were out of 200 tumor cells per random high-power field. In all eight considered candidate focal amplifications. Of 463 GBM profiles cases examined, FISH confirmed that focal CNA, regardless examined, focal PDGFRA-region amplification was found in 74 of amplitude, signified high-level RTK amplification in a sub- (16%). In many of these cases, PDGFRA was either the only RTK population of cells. Overall, EGFR amplification was seen in 40– in the amplicon or was the only RTK spanned by the peak of the 70% of cells and PDGFRA amplification in 10–30% (Fig. 2). In all CNA (n = 29, 39%). In contrast, KIT and KDR were never in- cases examined, the pattern of amplification appeared to be ei- dependently amplified. Most cases showed amplification equally ther exclusively or predominantly of extrachromosomal double across PDGFRA and KIT (n = 17) or PDGFRA, KIT, and KDR minute form. (n = 28). Because PDGFRA is the only RTK consistently am- EGFR and PDGFRA amplification was then assessed by dual- plified in the locus and is a known target of mutation in GBM (16), color FISH to determine the overlap in distribution. As shown in we refer to PDGFRA-region amplification simply as PDGFRA Fig. 2, all tumors demonstrated a common pattern in which the amplification. Focal amplification of EGFR was found in 214 majority of cells showed amplification of a single RTK (either samples (46%) and of MET in 12 samples (2.6%). EGFR or PDGFRA). The fraction of cells harboring amplification Coamplifications of EGFR, MET, and/or PDGFRA were ob- of both receptors ranged from 0 to 28% but comprised a minority served in 34 samples (7.3% of TCGA GBM). Remarkably, 43% of of the population in all cases. Examples of dual-color FISH PDGFRA-amplified tumors demonstrated coamplification of ei- are shown in Fig. 3. PDGFRA-amplified and PDGFRA/EGFR- ther EGFR or MET.Ofthe12MET-amplified tumors, 3 had EGFR coamplified cells were typically broadly distributed throughout and 5 had PDGFRA amplification (including one case with both). the tumors and were in all cases interspersed with EGFR-ampli- Fig. 1A summarizes the incidence of focal CNA found among the fied and nonamplified cells (example in Fig. S2). All tumors three RTKs considered. Focal CNA involving PDGFRA and/or demonstrated regions without PDGFRA-amplified cells (i.e., EGFR typically reached log2 ratios above 2, implying an average of EGFR-amplified only) but the converse, a region with exclusive more than eight gene copies per cell (Fig. 1B). PDGFRA-amplified cells, was not seen in any sample. In many coamplified cases the locus log2 ratios were below 2 and in the range commonly associated with gains of broad chro- Genomic Characteristics of RTK-Coamplified Tumors. Analysis of mosomal regions or chromosomal arms. However, the high GTS TCGA genomic data found no distinguishing characteristics of focality scores rule out broad CNA and suggest instead that focal dual RTK coamplified tumors compared with other GBMs.