Expression of Interleukin-13 Receptor A2 in Glioblastoma Multiforme: Implications for Targeted Therapies

Priority Report

John S. Jarboe,1 Kory R. Johnson,2 Yong Choi,1 Russell R. Lonser,3 and John K. Park1

1Surgical and Molecular Neuro-oncology Unit, 2Bioinformatics Group, Intramural Information Technology Program, Division of Intramural Research, and 3Surgical Neurology Branch, National Institute of Neurological Disorders and Stroke, NIH, Bethesda, Maryland

Abstract quantities of IL4, an effect referred to as IL4-independent IL13 Glioblastoma multiforme is the most common primary binding (2–5). The cell surface expression of a monomeric 42-kDa malignant brain tumor and despite treatment with surgery, receptor capable of binding IL13 but not IL4, termed IL13 receptor a2 (IL13Ra2), was used to explain this effect (6). It was also initially radiation, and chemotherapy, the median survival of patients a with glioblastoma multiforme is f1 year. Glioblastoma multi- reported that significant IL13R 2 expression, as assessed by Northern blotting, occurs exclusively in the testes and in malignant forme explants and cell lines have been reported to over- a express the interleukin-13 receptor A2 subunit (IL13RA2) glioma tissues (7). The putative relative restriction of IL13R 2 relative to nonneoplastic brain. Based on this finding, a expression to glioma cells was subsequently used as a rationale for recombinant cytotoxin composed of IL13 ligand and a trun- the development of several IL13-based treatment strategies including a recombinant cytotoxin composed of IL13 and a cated form of Pseudomonas aeruginosa exotoxin A (IL13- PE38QQR) was developed for the targeted treatment of truncated form of Pseudomonas aeruginosa exotoxin A (IL13- glioblastoma multiforme tumors. In a recently completed PE38QQR; ref. 8). IL13-PE38QQR internalized by cells expressing phase III clinical trial, however, IL13-PE38QQR was found to IL13 receptor complexes enzymatically inhibits protein synthesis be no more effective than an existing therapy in prolonging and causes apoptotic cell death (9). The IC50 of IL13-PE38QQR survival. To determine possible explanations for this result, in IL13 receptor–expressing cells has been reported to be as low as we analyzed the relative expression levels of IL13RA2in 0.1 ng/mL (8). A phase III clinical trial assessing the efficacy of IL13- glioblastoma multiforme and nonneoplastic brain specimens PE38QQR administered intratumorally via convection-enhanced using publicly available oligonucleotide microarray data- delivery has been completed recently, but detailed results have not bases, quantitative real-time reverse transcription PCR, and as yet been published. NeoPharm, the study sponsor, has however released preliminary information indicating that the median immunohistochemical staining. Increased expression of the IL13Ra2 gene relative to nonneoplastic brain was observed survival of patients treated with IL13-PE38QQR was 36.4 weeks, in 36 of 81 (44%) and 8 of 17 (47%) tumor specimens by whereas that of patients treated with a slow-release carmustine microarray and quantitative real-time reverse transcription wafer was 35.3 weeks (10). To identify factors that may have influenced the outcomes of IL13-PE38QQR–treated patients, we PCR analyses, respectively. Immunohistochemical staining of a tumor specimens showed highly variable expression of analyzed the relative expression levels of IL13R 2 in glioblastoma IL13RA2 protein both within and across specimens. These multiforme and nonneoplastic brain specimens. Using two data indicate that prescreening of subjects may be of benefit independent methods and two independent sets of patient speci- A mens, we determined that the percentage of glioblastoma multi- in future trials of IL13R 2 targeting therapies. [Cancer Res a 2007;67(17):7983–6] forme tumors with relative overexpression of IL13R 2 is <50%. The relevance of our findings on the analysis of completed, as well as the design of future, IL13Ra2 targeting clinical trials is discussed. Introduction A highly sought after treatment for glioblastoma multiforme is one that eradicates neoplastic cells while sparing normal brain Materials and Methods tissues. A cell surface antigen differentially overexpressed by Microarray analysis. The Oncomine Web site4 (11) was used to examine glioblastoma multiforme cells could serve as a therapeutic target the differential expression of IL13Ra2 in brain tissues. The microarray data and the basis for such a treatment. Interleukin 4 (IL4) and of Sun et al. (12) was identified as an appropriate data set for the detailed interleukin 13 (IL13) are two immune regulatory cytokines that can comparison of IL13Ra2 gene expression in glioblastoma multiforme and nonneoplastic brain specimens and could be downloaded from the National compete for binding to a heterodimeric cell surface receptor 5 consisting of a 140-kDa IL4 receptor h subunit and a 45-kDa IL13 Center for Biotechnology Information Gene Expression Omnibus as record GDS1962. All statistical analyses were done using the program R-2.4.1.6 receptor a1 subunit (1). In contrast to most normal body tissues, Examination of the data by covariance-based principal component analysis high-grade gliomas can also bind IL13 in the presence of excess and Pearson’s correlation analysis was used to confirm the suitability of this data set for investigation of IL13Ra2 gene expression. The differential expression of IL13Ra2 between glioblastoma multiforme and nonneoplastic brain specimens was examined by Tukey box plot, XY scatterplot, and Note: Supplementary data for this article are available at Cancer Research Online density plot. (http://cancerres.aacrjournals.org/). Requests for reprints: John K. Park, Surgical and Molecular Neuro-oncology Unit, National Institute of Neurological Disorders and Stroke, NIH, Room 2B-1002, 35 Convent Drive, MSC 3706, Bethesda, MD 20892. Phone: 301-402-6935; Fax: 301-480- 0099; E-mail: [email protected]. 4 http://www.oncomine.org I2007 American Association for Cancer Research. 5 http://www.ncbi.nlm.nih.gov/geo doi:10.1158/0008-5472.CAN-07-1493 6 http://www.r-project.org www.aacrjournals.org 7983 Cancer Res 2007; 67: (17). September 1, 2007

Quantitative real-time reverse transcription PCR. Seventeen glio- plot was generated and confirmed that the distribution of expres- blastoma multiforme and six nonneoplastic brain tissue samples, obtained sion of IL13Ra2 among glioblastoma multiforme samples and via neurosurgical resection in accordance with the NIH (NIH, Bethesda, nonneoplastic brain samples is overlapping and mixed (Fig. 1C). MD) institutional review board–approved human tissue collection proto- Quantitative real-time reverse transcription PCR. To confirm cols, were randomly selected for study. Informed consent for tissue the oligonucleotide microarray finding that less than half of collection was obtained from each subject. Total RNA was extracted from a snap-frozen tissues using Trizol reagent (Invitrogen). The RNA was treated glioblastoma multiforme samples have overexpression of IL13R 2, with DNase I (Invitrogen) to eliminate traces of genomic DNA and cDNAs we used both a different set of patient samples and a different were synthesized using SuperScript II Reverse Transcriptase (Invitrogen) method, quantitative real-time reverse transcription PCR (QRT- and random primers (Invitrogen). All procedures were done according to PCR). QRT-PCR analyses of 17 glioblastoma multiforme and 6 the manufacturer’s protocols. The integrity of the cDNA template was nonneoplastic brain samples were done (Fig. 2). The mean IL13Ra2 verified by standard PCR amplification of the human 18S rRNA product at expression level of the six nonneoplastic brain specimens was an annealing temperature of 50jC for 15 cycles. The primer sequences for assigned an arbitrary value of 1.0 and the fold increase in expres- ¶ ¶ the 18S rRNA product were 5 -GGAATAATGGAATAGGACC-3 (sense) and sion for each of the glioblastoma multiforme specimens relative to ¶ ¶ 5 -GCTCCACCAACTAAGAAC-3 (antisense). Quantitative PCR was carried this mean was determined. Individual glioblastoma multiforme out using FastStart SYBR Green Master (Roche) in the Prism 7900HT samples were considered to have significantly different expression sequence detection system (Applied Biosystems) for 40 cycles. The primers a for amplification of IL13Ra2were5¶-AATGGCTTTCGTTTGCTTGG-3¶ of IL13R 2 compared with nonneoplastic brain samples when the (sense) and 5¶-ACGCAATCCATATCCTGAAC-3¶ (antisense; 13). A cycle threshold value in the linear range of amplification was selected for each sample and normalized for level of 18S rRNA expression. The relative IL13Ra2 expression level of each sample was calculated using the formula DDCT 2 , where DDCT is the difference between the selected cycle threshold value of a particular sample and the mean of the cycle thresholds of the nonneoplastic brain samples (14). The mean IL13Ra2 expression level of the six nonneoplastic brain samples was assigned an expression value of 1.0 and the fold increase or decrease in IL13Ra2 expression was determined for each control and glioblastoma multiforme sample. Individual glioblastoma multiforme samples were considered to have significantly different expression of IL13Ra2 compared with nonneoplastic brain when the P value for a t test comparing the two was <0.05. Experiments were done in triplicate. Immunofluorescence histochemistry. Five-micrometer-thick paraffin- embedded sections of one nonneoplastic brain specimen and six glioblastoma multiforme specimens were dewaxed, rehydrated, and subjected to heat antigen retrieval with 10 mmol/L sodium citrate at 95jC for 20 min. The sections were then incubated with goat anti-IL13Ra2 polyclonal antibodies (1:500 dilution; R&DSystems) followed by Alexa Flour 647–conjugated donkey anti-goat antibodies (1:500 dilution; Molecular Probes). Sections were counterstained with 300 nmol/L 4¶,6-diamidino-2- phenylindole (DAPI; Sigma) and images were acquired using an LSM 510 confocal laser scanning microscope (Carl Zeiss, Inc.). To show the specificity of the goat anti-IL13Ra2 polyclonal antibodies, 293FT human embryonic kidney cells (Invitrogen) stably transfected with an IL13Ra2 cDNA (Origene) and nontransfected control 293FT cells were stained and imaged as described.

Results Microarray analysis. To determine the relative gene expression levels of IL13Ra2 in glioblastoma multiforme and nonneoplastic brain samples, we first analyzed a publicly available oligonucleotide microarray data set. Examination of the data set from the study by Figure 1. Analysis of oligonucleotide microarray data for IL13Ra2 gene Sun et al. (12) using covariance-based principal component expression. A, Tukey box plot comparing the natural log of the MAS5 expression analysis and Pearson’s correlation analysis indicated that this data values of IL13Ra2 in nonneoplastic brain control (green) and glioblastoma a multiforme (GBM; red) samples. Boxes, interquartile range of expression values. set was suitable for evaluation of IL13R 2 expression among glio- The whiskers extend to the highest and lowest values of expression that are not blastoma multiforme and nonneoplastic brain samples. A Tukey considered outliers. Circles, outliers. B, XY scatterplot of expression levels of box plot comparing glioblastoma multiforme samples to nonneo- individual samples. X axis, individual control (green) or glioblastoma multiforme (red) sample; Y axis, natural log of the MAS5 IL13Ra2 expression level of the plastic brain samples indicates that the ranges of expression of represented sample. The solid line indicates the mean expression level of the IL13Ra2 among these groups are overlapping and the distribution controls and the hashed line indicates two SDs above this value. Samples that lie above this line are considered to exhibit significantly greater expression of of expression among glioblastoma multiforme samples is skewed IL13Ra2 than the controls. C, sample density distribution plot comparing the (Fig. 1A). When this data is presented as an XY scatterplot, 36 of 81 natural log of the MAS5 expression levels of IL13Ra2 in nonneoplastic brain (44.4%) glioblastoma multiforme samples exhibit expression of control (green) and glioblastoma multiforme (red) samples. For a given IL13Ra2 a gene expression value (X axis), the relative density of samples is represented by IL13R 2 greater than two SDs from the expression mean for non- the density estimate (Y axis). For all panels, MAS5 Expression denotes the neoplastic brain samples (Fig. 1B). A sample density distribution expression values derived from the Affymetrix suite version 5 software.

Cancer Res 2007; 67: (17). September 1, 2007 7984 www.aacrjournals.org

binding sites (2–4), whereas nonquantitative RT-PCR analysis indicated that 14 of 17 (82%) tumors show expression of IL13Ra2 (15). Similarly, it has been reported that 11 of 11 glioblastoma multiforme specimens overexpress IL13Ra2 as determined by in situ hybridization and immunohistochemical staining (16). In contrast to these results, our analysis of publicly available oligonucleotide microarray data sets suggests that the percentage of glioblastoma multiforme tumors that overexpress IL13Ra2is 44% (36 of 81 specimens). In concordance with this latter result, the percentage of glioblastoma multiforme tumors that overexpress IL13Ra2 as determined by QRT-PCR analysis is 47% (8 of 17 specimens). The results presented here about the relative expression levels of IL13Ra2 in glioblastoma multiforme specimens compared with nonneoplastic brain tissue differ from those of previously published studies (15, 16). The discrepancies in the results may in part be accounted for by differences in the materials and methods used. Joshi et al. (15) used primary cell cultures derived from glioblas- Figure 2. QRT-PCR results for expression of IL13Ra2 in patient samples of glioblastoma multiforme (black columns) and nonneoplastic brain control toma multiforme and nonneoplastic brain explants, whereas we (white columns). Y axis, natural log of the fold increase in IL13Ra2 expression and Sun et al. (12) used actual tissue specimens. Previous studies level of a particular sample relative to the mean IL13Ra2 expression level have shown that the gene expression signatures of tumor tissues of the nonneoplastic brain samples. Horizontal solid line, mean IL13Ra2 expression of the nonneoplastic brain samples. Samples with fold expression can differ significantly from that of primary cultures derived from levels above the hashed line show significantly higher IL13Ra2 expression than those tissues (17). With regard to differences in methods, we used the mean expression level of the nonneoplastic brain samples. oligonucleotide microarrays and quantitative RT-PCR rather than nonquantitative RT-PCR (15) and in situ hybridization (16), which P value for a t test comparing the two was <0.05. Using this is also nonquantitative. criterion, 8 of 17 (47%) glioblastoma multiforme samples exhibited Although the results of a recent phase III trial assessing the increased IL13Ra2 expression relative to nonneoplastic brain efficacy of IL13-PE38QQR have yet to be published, a preliminary samples (Fig. 2). information release indicates that it is no more effective than Immunohistochemistry. As a third method for analyzing existing therapies. This is not unexpected given our finding that IL13Ra2 expression in glioblastoma multiforme tumors, we did less than half of tumors overexpress IL13Ra2. Additional factors immunohistochemical staining of glioblastoma multiforme and that may have contributed to the lack of efficacy of IL13-PE38QQR nonneoplastic brain paraffin-embedded sections. The relative spe- are suboptimal drug infusion technique in some patients (18) and cificity of the anti-IL13Ra2 antibodies used was first confirmed by the high variability of IL13Ra2 expression from region to region staining control and IL13Ra2 cDNA–transfected human embryonic within some tumors. Although the intratumoral heterogeneity of kidney–derived cell lines (Supplementary Fig. S1A and B). Immu- expression could be an experimental artifact due to inherent nohistochemical staining revealed no detectable IL13Ra2 protein variations in the fixation and immunohistochemical processing of expression in nonneoplastic brain (Fig. 3A). Specific membrane staining was observed in four of six glioblastoma multiforme specimens and the distribution of expression, when present, was highly variable from region to region within a single specimen. Two of the specimens that stained positively showed expression of IL13Ra2 throughout the section. The other two specimens showed mixed expression, with areas of high expression and clusters of neoplastic cells that were negative for IL13Ra2 expression. Photo- micrographs of representative sections showing high, mixed, and negative expression of IL13Ra2 protein are shown (Fig. 3B–D). These qualitative results confirm the quantitative results obtained using oligonucleotide microarray and QRT-PCR and also show the intratumoral heterogeneity of IL13Ra2 expression.

Discussion In principle, cytotoxins that recognize and bind specific cell surface proteins carry great promise for the treatment of tumors. The success of such treatments, however, is dependent on the Figure 3. IL13Ra2 expression as determined by immunohistochemical exclusive or enriched expression of the targeted protein on tumor staining. A to D, expression of IL13Ra2 in paraffin-embedded sections of or tumor-dependent cells. Previous reports have indicated that nonneoplastic brain white matter (A) and paraffin-embedded sections of IL13Ra2 is expressed by a vast majority of glioblastoma multiforme glioblastoma multiforme tumors (B–D). Representative glioblastoma multiforme tumor sections with high (B), mixed (C), and negative (D) IL13Ra2 explants. Autoradiography of glioblastoma multiforme specimens expression. Anti-IL13Ra2 antibody staining (green) and DAPI staining of nuclei indicated that nearly all tumors possess IL4-independent IL13 (blue). Bar,50Am. www.aacrjournals.org 7985 Cancer Res 2007; 67: (17). September 1, 2007

Downloaded from cancerres.aacrjournals.org on October 3, 2021. © 2007 American Association for Cancer Research. Cancer Research clinical tissues or to expression levels of IL13Ra2 below the available patient samples for the expression of IL13Ra2 and a detection limit of the antibodies used, this heterogeneity has been stratification of patients by degree and or pattern of tumor reported by others as well (16). This is of potential clinical expression. The data obtained from such retrospective analyses, significance as the effect of IL13-PE38QQR, the induction of although problematic for determining the success of already apoptosis, is reported to be highly specific to IL13Ra2-expressing completed trials, could be used in the improved planning of future cells (9). The IL13Ra2-expressing fraction of cells within an trials. At a minimum, demonstration of tumor IL13Ra2 expression IL13Ra2-expressing tumor may therefore respond to IL13- should be an eligibility criterion for any such trials. In this way, PE38QQR, but the IL13Ra2-negative fraction will be unresponsive patients with a higher likelihood of benefiting from the therapy can and continue to proliferate. This could serve to explain any partial be considered for enrollment, whereas those with a lower or temporary responses to IL13-PE38QQR that may have occurred. likelihood can be encouraged to pursue other options. Also of interest is the incidence and nature of any non–central nervous system toxicities that may have occurred during the trial. Although IL13Ra2 has been described as a cancer/testis Acknowledgments antigen (7), its reported expression in the kidney, spleen, liver, lung, Received 4/23/2007; revised 6/26/2007; accepted 7/17/2007. Grant support: Intramural Research Program of the NIH, National Institute of thymus, respiratory epithelium, and monocytes (1, 19) indicates Neurological Disorders and Stroke. that it is more likely a tumor-associated or tumor-overexpressed The costs of publication of this article were defrayed in part by the payment of page antigen. charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Rather than dismiss the further testing and potential use of We thank A. Sedlock for providing technical assistance and G. Park for critical IL13-PE38QQR, we recommend a retrospective analysis of all review of the manuscript.

References of restrictive receptor for interleukin 13, a brain tumor- 14. Dussault AA, Pouliot M. Rapid and simple compar- associated cancer/testis antigen. Mol Med 2000;6:440–9. ison of messenger RNA levels using real-time PCR. Biol 1. Hershey GK. IL-13 receptors and signaling pathways: 8. Debinski W, Obiri NI, Powers SK, Pastan I, Puri RK. Proced Online 2006;8:1–10. an evolving web. J Allergy Clin Immunol 2003;111:677–90. Human glioma cells overexpress receptors for interleu- 15. Joshi BH, Plautz GE, Puri RK. Interleukin-13 receptor 2. Debinski W, Gibo DM, Hulet SW, Connor JR, Gillespie kin 13 and are extremely sensitive to a novel chimeric a chain: a novel tumor-associated transmembrane GY. Receptor for interleukin 13 is a marker and protein composed of interleukin 13 and Pseudomonas protein in primary explants of human malignant therapeutic target for human high-grade gliomas. Clin exotoxin. Clin Cancer Res 1995;1:1253–8. gliomas. Cancer Res 2000;60:1168–72. Cancer Res 1999;5:985–90. 9. Kawakami M, Kawakami K, Puri RK. Intratumor ad- 16. Kawakami M, Kawakami K, Takahashi S, Abe M, 3. Debinski W, Gibo DM, Slagle B, Powers SK, Gillespie ministration of interleukin 13 receptor-targeted cytotoxin Puri RK. Analysis of interleukin-13 receptor a2 expres- GY. Receptor for interleukin 13 is abundantly and induces apoptotic cell death in human malignant glioma sion in human pediatric brain tumors. Cancer 2004;101: specifically over-expressed in patients with glioblastoma tumor xenografts. Mol Cancer Ther 2002;1:999–1007. 1036–42. multiforme. Int J Oncol 1999;15:481–6. 10. Japsen B. NeoPharm drug fails test: cancer therapy 17. Lee J, Kotliarova S, Kotliarov Y, et al. Tumor stem 4. Debinski W, Slagle B, Gibo DM, Powers SK, Gillespie bust sends stock into dive. Chicago Tribune. 2006 Dec cells derived from glioblastomas cultured in bFGF and GY. Expression of a restrictive receptor for interleukin 12; Sect. Business:1. EGF more closely mirror the phenotype and genotype of 13 is associated with glial transformation. J Neurooncol 11. Rhodes DR, Yu J, Shanker K, et al. ONCOMINE: a primary tumors than do serum-cultured cell lines. 2000;48:103–11. cancer microarray database and integrated data-mining Cancer Cell 2006;9:391–403. 5. Debinski W, Miner R, Leland P, Obiri NI, Puri RK. platform. Neoplasia 2004;6:1–6. 18. Sampson JH, Brady ML, Petry NA, et al. Intracerebral Receptor for interleukin (IL) 13 does not interact with 12. Sun L, Hui AM, Su Q, et al. Neuronal and glioma- infusate distribution by convection-enhanced delivery in IL4 but receptor for IL4 interacts with IL13 on human derived stem cell factor induces angiogenesis within the humans with malignant gliomas: descriptive effects of glioma cells. J Biol Chem 1996;271:22428–33. brain. Cancer Cell 2006;9:287–300. target anatomy and catheter positioning. Neurosurgery 6. Mintz A, Gibo DM, Slagle-Webb B, Christensen ND, 13. Murata T, Obiri NI, Debinski W, Puri RK. Structure of 2007;60:ONS89–98. Debinski W. IL-13Ra2 is a glioma-restricted receptor for IL-13 receptor: analysis of subunit composition in 19. Parry RG, Gillespie KM, Mathieson PW. Effects of interleukin-13. Neoplasia 2002;4:388–99. cancer and immune cells. Biochem Biophys Res type 2 cytokines on glomerular epithelial cells. Exp 7. Debinski W, Gibo DM. Molecular expression analysis Commun 1997;238:90–4. Nephrol 2001;9:275–83.

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Downloaded from cancerres.aacrjournals.org on October 3, 2021. © 2007 American Association for Cancer Research. Expression of Interleukin-13 Receptor α2 in Glioblastoma Multiforme: Implications for Targeted Therapies

John S. Jarboe, Kory R. Johnson, Yong Choi, et al.

Cancer Res 2007;67:7983-7986.

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