Top

Published in:

Open Access 01-05-2014 | Original Paper

Quantitative assessment of intragenic receptor tyrosine kinase deletions in primary glioblastomas: their prevalence and molecular correlates

Authors: Edward R. Kastenhuber, Jason T. Huse, Samuel H. Berman, Alicia Pedraza, Jianan Zhang, Yoshiyuki Suehara, Agnes Viale, Magali Cavatore, Adriana Heguy, Nicholas Szerlip, Marc Ladanyi, Cameron W. Brennan

Published in: Acta Neuropathologica | Issue 5/2014

Abstract

Intragenic deletion is the most common form of activating mutation among receptor tyrosine kinases (RTK) in glioblastoma. However, these events are not detected by conventional DNA sequencing methods commonly utilized for tumor genotyping. To comprehensively assess the frequency, distribution, and expression levels of common RTK deletion mutants in glioblastoma, we analyzed RNA from a set of 192 glioblastoma samples from The Cancer Genome Atlas for the expression of EGFRvIII, EGFRvII, EGFRvV (carboxyl-terminal deletion), and PDGFRAΔ8,9. These mutations were detected in 24, 1.6, 4.7, and 1.6 % of cases, respectively. Overall, 29 % (55/189) of glioblastomas expressed at least one RTK intragenic deletion transcript in this panel. For EGFRvIII, samples were analyzed by both quantitative real-time PCR (QRT-PCR) and single mRNA molecule counting on the Nanostring nCounter platform. Nanostring proved to be highly sensitive, specific, and linear, with sensitivity comparable or exceeding that of RNA seq. We evaluated the prognostic significance and molecular correlates of RTK rearrangements. EGFRvIII was only detectable in tumors with focal amplification of the gene. Moreover, we found that EGFRvIII expression was not prognostic of poor outcome and that neither recurrent copy number alterations nor global changes in gene expression differentiate EGFRvIII-positive tumors from tumors with amplification of wild-type EGFR. The wide range of expression of mutant alleles and co-expression of multiple EGFR variants suggests that quantitative RNA-based clinical assays will be important for assessing the relative expression of intragenic deletions as therapeutic targets and/or candidate biomarkers. To this end, we demonstrate the performance of the Nanostring assay in RNA derived from routinely collected formalin-fixed paraffin-embedded tissue.

Available only for authorised users

Al-Nedawi K, Meehan B, Micallef J et al (2008) Intercellular transfer of the oncogenic receptor EGFRvIII by microvesicles derived from tumour cells. Nat Cell Biol 10:619–624. doi:10.1038/ncb1725 PubMedCrossRef

Aldape KD, Ballman K, Furth A et al (2004) Immunohistochemical detection of EGFRvIII in high malignancy grade astrocytomas and evaluation of prognostic significance. J Neuropathol Exp Neurol 63:700–707PubMed

Biernat W, Huang H, Yokoo H, Kleihues P, Ohgaki H (2004) Predominant expression of mutant EGFR (EGFRvIII) is rare in primary glioblastomas. Brain Pathol 14:131–136PubMedCrossRef

Brennan C, Momota H, Hambardzumyan D et al (2009) Glioblastoma subclasses can be defined by activity among signal transduction pathways and associated genomic alterations. PLoS One 4:e7752. doi:10.1371/journal.pone.0007752 PubMedCentralPubMedCrossRef

Brennan CW, Verhaak RG, McKenna A et al (2013) The somatic genomic landscape of glioblastoma. Cell 155:462–477. doi:10.1016/j.cell.2013.09.034 PubMedCrossRef

Chen WS, Lazar CS, Lund KA et al (1989) Functional independence of the epidermal growth factor receptor from a domain required for ligand-induced internalization and calcium regulation. Cell 59:33–43PubMedCrossRef

Cho J, Pastorino S, Zeng Q et al (2011) Glioblastoma-derived epidermal growth factor receptor carboxyl-terminal deletion mutants are transforming and are sensitive to EGFR-directed therapies. Cancer Res 71:7587–7596. doi:10.1158/0008-5472.CAN-11-0821 PubMedCentralPubMedCrossRef

Dunn GP, Rinne ML, Wykosky J et al (2012) Emerging insights into the molecular and cellular basis of glioblastoma. Genes Dev 26:756–784. doi:10.1101/gad.187922.112 PubMedCentralPubMedCrossRef

Ekstrand AJ, Sugawa N, James CD, Collins VP (1992) Amplified and rearranged epidermal growth factor receptor genes in human glioblastomas reveal deletions of sequences encoding portions of the N- and/or C-terminal tails. Proc Natl Acad Sci USA 89:4309–4313PubMedCentralPubMedCrossRef

10.

Frederick L, Wang XY, Eley G, James CD (2000) Diversity and frequency of epidermal growth factor receptor mutations in human glioblastomas. Cancer Res 60:1383–1387PubMed

11.

Gajadhar AS, Bogdanovic E, Munoz DM, Guha A (2012) In Situ analysis of mutant EGFRs prevalent in glioblastoma multiforme reveals aberrant dimerization, activation, and differential response to anti-EGFR targeted therapy. Mol Cancer Res 10:428–440. doi:10.1158/1541-7786.MCR-11-0531 PubMedCrossRef

12.

Gan HK, Kaye AH, Luwor RB (2009) The EGFRvIII variant in glioblastoma multiforme. J Clin Neurosci 16:748–754. doi:10.1016/j.jocn.2008.12.005 PubMedCrossRef

13.

Geiss GK, Bumgarner RE, Birditt B et al (2008) Direct multiplexed measurement of gene expression with color-coded probe pairs. Nat Biotechnol 26:317–325. doi:10.1038/nbt1385 PubMedCrossRef

14.

Grandal MV, Zandi R, Pedersen MW, Willumsen BM, van Deurs B, Poulsen HS (2007) EGFRvIII escapes down-regulation due to impaired internalization and sorting to lysosomes. Carcinogenesis 28:1408–1417. doi:10.1093/carcin/bgm058 PubMedCrossRef

15.

Heimberger AB, Hlatky R, Suki D et al (2005) Prognostic effect of epidermal growth factor receptor and EGFRvIII in glioblastoma multiforme patients. Clin Cancer Res 11:1462–1466. doi:10.1158/1078-0432.CCR-04-1737 PubMedCrossRef

16.

Horvath S, Zhang B, Carlson M et al (2006) Analysis of oncogenic signaling networks in glioblastoma identifies ASPM as a molecular target. Proc Natl Acad Sci USA 103:17402–17407. doi:10.1073/pnas.0608396103 PubMedCentralPubMedCrossRef

17.

Huang PH, Mukasa A, Bonavia R et al (2007) Quantitative analysis of EGFRvIII cellular signaling networks reveals a combinatorial therapeutic strategy for glioblastoma. Proc Natl Acad Sci USA 104:12867–12872. doi:10.1073/pnas.0705158104 PubMedCentralPubMedCrossRef

18.

Huse JT, Holland E, Deangelis LM (2013) Glioblastoma: molecular analysis and clinical implications. Annu Rev Med 64:59–70. doi:10.1146/annurev-med-100711-143028 PubMedCrossRef

19.

Inda MM, Bonavia R, Mukasa A et al (2010) Tumor heterogeneity is an active process maintained by a mutant EGFR-induced cytokine circuit in glioblastoma. Genes Dev 24:1731–1745. doi:10.1101/gad.1890510 PubMedCentralPubMedCrossRef

20.

Kuan CT, Wikstrand CJ, Bigner DD (2001) EGF mutant receptor vIII as a molecular target in cancer therapy. Endocr Relat Cancer 8:83–96PubMedCrossRef

21.

Kumabe T, Sohma Y, Kayama T, Yoshimoto T, Yamamoto T (1992) Amplification of alpha-platelet-derived growth factor receptor gene lacking an exon coding for a portion of the extracellular region in a primary brain tumor of glial origin. Oncogene 7:627–633PubMed

22.

Lal A, Glazer CA, Martinson HM et al (2002) Mutant epidermal growth factor receptor up-regulates molecular effectors of tumor invasion. Cancer Res 62:3335–3339PubMed

23.

Lee JC, Vivanco I, Beroukhim R et al (2006) Epidermal growth factor receptor activation in glioblastoma through novel missense mutations in the extracellular domain. PLoS Med 3:e485. doi:10.1371/journal.pmed.0030485 PubMedCentralPubMedCrossRef

24.

Liu L, Backlund LM, Nilsson BR et al (2005) Clinical significance of EGFR amplification and the aberrant EGFRvIII transcript in conventionally treated astrocytic gliomas. J Mol Med 83:917–926. doi:10.1007/s00109-005-0700-2 PubMedCentralPubMedCrossRef

25.

Luwor RB, Zhu HJ, Walker F et al (2004) The tumor-specific de2-7 epidermal growth factor receptor (EGFR) promotes cells survival and heterodimerizes with the wild-type EGFR. Oncogene 23:6095–6104. doi:10.1038/sj.onc.1207870 PubMedCrossRef

26.

Mellinghoff IK, Wang MY, Vivanco I et al (2005) Molecular determinants of the response of glioblastomas to EGFR kinase inhibitors. N Engl J Med 353:2012–2024. doi:10.1056/NEJMoa051918 PubMedCrossRef

27.

Mellinghoff IK, Schultz N, Mischel PS, Cloughesy TF (2012) Will kinase inhibitors make it as glioblastoma drugs? Curr Top Microbiol Immunol 355:135–169. doi:10.1007/82_2011_178 PubMedCentralPubMed

28.

Mitsudomi T, Yatabe Y (2010) Epidermal growth factor receptor in relation to tumor development: EGFR gene and cancer. FEBS J 277:301–308. doi:10.1111/j.1742-4658.2009.07448.x PubMedCrossRef

29.

Mizoguchi M, Betensky RA, Batchelor TT, Bernay DC, Louis DN, Nutt CL (2006) Activation of STAT3, MAPK, and AKT in malignant astrocytic gliomas: correlation with EGFR status, tumor grade, and survival. J Neuropathol Exp Neurol 65:1181–1188. doi:10.1097/01.jnen.0000248549.14962.b2 PubMedCrossRef

30.

Nagane M, Coufal F, Lin H, Bogler O, Cavenee WK, Huang HJ (1996) A common mutant epidermal growth factor receptor confers enhanced tumorigenicity on human glioblastoma cells by increasing proliferation and reducing apoptosis. Cancer Res 56:5079–5086PubMed

31.

Network T (2008) Comprehensive genomic characterization defines human glioblastoma genes and core pathways. Nature 455:1061–1068. doi:10.1038/nature07385 CrossRef

32.

Nicholas MK, Lukas RV, Jafri NF, Faoro L, Salgia R (2006) Epidermal growth factor receptor––mediated signal transduction in the development and therapy of gliomas. Clin Cancer Res 12:7261–7270. doi:10.1158/1078-0432.CCR-06-0874 PubMedCrossRef

33.

Nishikawa R, Sugiyama T, Narita Y, Furnari F, Cavenee WK, Matsutani M (2004) Immunohistochemical analysis of the mutant epidermal growth factor, deltaEGFR, in glioblastoma. Brain Tumor Pathol 21:53–56PubMedCrossRef

34.

Noushmehr H, Weisenberger DJ, Diefes K et al (2010) Identification of a CpG island methylator phenotype that defines a distinct subgroup of glioma. Cancer Cell 17:510–522. doi:10.1016/j.ccr.2010.03.017 PubMedCentralPubMedCrossRef

35.

Okada H, Kohanbash G, Zhu X et al (2009) Immunotherapeutic approaches for glioma. Crit Rev Immunol 29:1–42PubMedCentralPubMedCrossRef

36.

Ozawa T, Brennan CW, Wang L et al (2010) PDGFRA gene rearrangements are frequent genetic events in PDGFRA-amplified glioblastomas. Genes Dev 24:2205–2218. doi:10.1101/gad.1972310 PubMedCentralPubMedCrossRef

37.

Phillips HS, Kharbanda S, Chen R et al (2006) Molecular subclasses of high-grade glioma predict prognosis, delineate a pattern of disease progression, and resemble stages in neurogenesis. Cancer Cell 9:157–173. doi:10.1016/j.ccr.2006.02.019 PubMedCrossRef

38.

Pines G, Huang PH, Zwang Y, White FM, Yarden Y (2010) EGFRvIV: a previously uncharacterized oncogenic mutant reveals a kinase auto-inhibitory mechanism. Oncogene 29:5850–5860. doi:10.1038/onc.2010.313 PubMedCentralPubMedCrossRef

39.

Prigent SA, Nagane M, Lin H et al (1996) Enhanced tumorigenic behavior of glioblastoma cells expressing a truncated epidermal growth factor receptor is mediated through the Ras-Shc-Grb2 pathway. J Biol Chem 271:25639–25645PubMedCrossRef

40.

Rand V, Huang J, Stockwell T et al (2005) Sequence survey of receptor tyrosine kinases reveals mutations in glioblastomas. Proc Natl Acad Sci USA 102:14344–14349. doi:10.1073/pnas.0507200102 PubMedCentralPubMedCrossRef

41.

Smyth GK (2004) Linear models and empirical bayes methods for assessing differential expression in microarray experiments. Stat Appl Genet Mol Biol 3: Article3 Doi: 10.2202/1544-6115.1027

42.

Sugawa N, Ekstrand AJ, James CD, Collins VP (1990) Identical splicing of aberrant epidermal growth factor receptor transcripts from amplified rearranged genes in human glioblastomas. Proc Natl Acad Sci USA 87:8602–8606PubMedCentralPubMedCrossRef

43.

Szerlip NJ, Pedraza A, Chakravarty D et al (2012) Intra-tumoral heterogeneity of receptor tyrosine kinases EGFR and PDGFRA amplification in glioblastoma defines subpopulations with distinct growth factor response. Proc Natl Acad Sci USA 109:3041–3046. doi:10.1073/pnas.1114033109 PubMedCentralPubMedCrossRef

44.

TCGA (2008) Comprehensive genomic characterization defines human glioblastoma genes and core pathways. Nature 455:1061–1068. doi:10.1038/nature07385 CrossRef

45.

Verhaak RG, Hoadley KA, Purdom E et al (2010) Integrated genomic analysis identifies clinically relevant subtypes of glioblastoma characterized by abnormalities in PDGFRA, IDH1, EGFR, and NF1. Cancer Cell 17:98–110. doi:10.1016/j.ccr.2009.12.020 PubMedCentralPubMedCrossRef

46.

Vivanco I, Robins HI, Rohle D et al (2012) Differential sensitivity of glioma- versus lung cancer-specific EGFR mutations to EGFR kinase inhibitors. Cancer Discov 2:458–471. doi:10.1158/2159-8290.CD-11-0284 PubMedCentralPubMedCrossRef

47.

Voldborg BR, Damstrup L, Spang-Thomsen M, Poulsen HS (1997) Epidermal growth factor receptor (EGFR) and EGFR mutations, function and possible role in clinical trials. Ann Oncol 8:1197–1206PubMedCrossRef

48.

Wells A, Welsh JB, Lazar CS, Wiley HS, Gill GN, Rosenfeld MG (1990) Ligand-induced transformation by a non-internalizing epidermal growth factor receptor. Science 247:962–964PubMedCrossRef

49.

Wiedemeyer R, Brennan C, Heffernan TP et al (2008) Feedback circuit among INK4 tumor suppressors constrains human glioblastoma development. Cancer Cell 13:355–364. doi:10.1016/j.ccr.2008.02.010 PubMedCentralPubMedCrossRef

50.

Wu JL, Abe T, Inoue R, Fujiki M, Kobayashi H (2004) IkappaBalphaM suppresses angiogenesis and tumorigenesis promoted by a constitutively active mutant EGFR in human glioma cells. Neurol Res 26:785–791. doi:10.1179/016164104225014139 PubMedCrossRef

51.

Yoshimoto K, Dang J, Zhu S et al (2008) Development of a real-time RT-PCR assay for detecting EGFRvIII in glioblastoma samples. Clin Cancer Res 14:488–493. doi:10.1158/1078-0432.CCR-07-1966 PubMedCrossRef

Title: Quantitative assessment of intragenic receptor tyrosine kinase deletions in primary glioblastomas: their prevalence and molecular correlates
Authors: Edward R. Kastenhuber
Jason T. Huse
Samuel H. Berman
Alicia Pedraza
Jianan Zhang
Yoshiyuki Suehara
Agnes Viale
Magali Cavatore
Adriana Heguy
Nicholas Szerlip
Marc Ladanyi
Cameron W. Brennan
Publication date: 01-05-2014
Publisher: Springer Berlin Heidelberg
Published in: Acta Neuropathologica / Issue 5/2014
Print ISSN: 0001-6322
Electronic ISSN: 1432-0533
DOI: https://doi.org/10.1007/s00401-013-1217-3

At a glance: The STEP trials

Springer Medicine

Quantitative assessment of intragenic receptor tyrosine kinase deletions in primary glioblastomas: their prevalence and molecular correlates

Abstract

At a glance: The STEP trials

Springer Medicine

Abstract

Please log in to get access to this content

Other articles of this Issue 5/2014

Acknowledgement to referees

Amyloidogenic α-synuclein seeds do not invariably induce rapid, widespread pathology in mice

Chemokine-mediated redirection of T cells constitutes a critical mechanism of glucocorticoid therapy in autoimmune CNS responses

P-glycoprotein regulates trafficking of CD8+ T cells to the brain parenchyma

Myopathy in Marinesco–Sjögren syndrome links endoplasmic reticulum chaperone dysfunction to nuclear envelope pathology

CSF Apo-E levels associate with cognitive decline and MRI changes