Longitudinal Expression Analysis of >v Integrins inHuman Gliomas Reveals Upregulation of
Integrin >vA3 as a Negative Prognostic Factor
Jens Schittenhelm, MD, Esther I. Schwab, CM, Jan Sperveslage, PhD, Marcos Tatagiba, MD,Richard Meyermann, MD, Falko Fend, MD, Simon L. Goodman, PhD, and Bence Sipos, MD
AbstractIntegrin inhibitors targeting >v series integrins are being tested for
their therapeutic potential in patients with brain tumors, but patho-logic studies have been limited by lack of antibodies suitable forimmunohistochemistry (IHC) on formalin-fixed, paraffin-embeddedspecimens. We compared the expression of >v integrins by IHC inbrain tumor and normal human brain samples with gene expressiondata in a public database using new rabbit monoclonal antibodiesagainst >vA3, >vA5, >vA6, and >vA8 complexes using both manualand automated microscopy analyses. Glial tumors usually shared an>vA3-positive/>vA5-positive/>vA8-positive/>vA6-negative phenotype.In 94 WHO (World Health Organization) grade II astrocytomas, 85anaplastic astrocytomas WHO grade III, and 324 glioblastomas fromarchival sources, expression of integrins generally increased with gradeof malignancy. Integrins >vA3 and >vA5 were expressed in many gli-oma vessels; the intensity of vascular expression of >vA3 increased withgrade of malignancy, whereas >vA8 was absent. Analysis of gene ex-pression in an independent cohort showed a similar increase in integrinexpression with tumor grade, particularly of ITGB3 and ITGB8; ITGB6was not expressed, consistent with the IHC data. Parenchymal >vA3expression and ITGB3 gene overexpression in glioblastomas were as-sociated with a poor prognosis, as revealed by survival analysis (Kaplan-Meier logrank, p = 0.016). Together, these data strengthen the rationalefor anti-integrin treatment of glial tumors.
Astrocytic brain tumors often recur because of theirrapid invasion and extensive migration into the surroundingtissue. The cell-cell and extracellular matrix (ECM)Ycell in-
teractions in these tumors that drive these processes aremediated in part by a group of integrin transmembrane recep-tors (1, 2). These proteins are obligate >/A heterodimers; eachchain has a large extracellular domain and, with the exceptionof >vA4, a short cytoplasmic domain. Integrin ligation initi-ates signals that activate cytoplasmic kinase cascades, thus reg-ulating diverse biologic functions, including cell attachment,differentiation, migration, wound healing, growth, and survival;hence, they play fundamental roles in many neoplasms (2Y4).To date, 24 integrin heterodimers have been identified, at least10 of which can be expressed by 1 cell type (5, 6). Five in-tegrins contain the >v subunit (>vA1, >vA3, >vA5, >vA6, and>vA8), suggesting a pivotal role for the >v subfamily (5, 7).Chemokines and growth factors can modulate cellular integrinexpression. Expression of >v complexes is often altered in tu-mors, and the expression pattern varies with the tumor type(8Y10). The >v integrins bind to Arg-Gly-Asp (RGD) sequencesin members of the provisional ECM in tumors (11, 12).
Integrins >vA3 and >vA5 are expressed in endothelialcells and tumor parenchyma of several types of cancer, sug-gesting a role in tumor initiation and progression (10, 12). In-tegrin A3-knockout mice display enhanced tumor growth and aproangiogenic phenotype (6, 13), which has led to the conceptthat >vA3 expression may mediate a balance between protu-mor and antitumor effects (14). Expression of >vA3 has beenreported in neoplastic tumor cells and in brain tumor vascula-ture (15, 16). The ECM protein osteopontin, which is oftenoverexpressed in gliomas (17), is a ligand of >vA3 (18). It isnoteworthy that integrins are overexpressed in high-grade gli-omas (15, 19, 20) but seem to be absent in low-grade tumors(20, 21). Inhibitory monoclonal antibodies and low-molecular-weight synthetic inhibitors of >v integrins are in clinical trials(22, 23), and mixed >vA3/>vA5 inhibitors can inhibit thegrowth of orthotopic brain tumor models (24Y26). The >v in-tegrin inhibitors may also reduce tumor vascular burden andenhance the effects of other therapeutic modalities such as ra-diotherapy (24, 27, 28).
Diffuse astrocytomas and glioblastomas (GBMs), themost common glial neoplasms, are most often located in thesubcortical or deep white matter of the cerebral hemispheres(29). Most gliomas develop de novo as primary GBMs, buttumors initially diagnosed as diffuse astrocytomas tend to prog-ress to a more malignant phenotype within 6 to 8 years, end-ing finally as genetically distinct secondary GBMs (30). Such
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Departments of Neuropathology (JSc, EIS, RM) and Pathology (JSp, FF, BS),Institute of Pathology and Neuropathology, and Department of Neuro-surgery (MT), University of Tubingen, Tubingen, Germany; and Depart-ment of Cellular Pharmacology-Oncology Platform (SLG), Merck KGaA,Darmstadt, Germany.
Send correspondence and reprint requests to: Jens Schittenhelm, MD, Depart-ment of Neuropathology, Institute of Pathology and Neuropathology, Uni-versity Tubingen, Calwerstr. 3, D-72076 Tubingen, Germany; E-mail: [email protected]
Jens Schittenhelm is supported by a grant from the Ludwig-Hiermaier Foun-dation for Applied Cancer Research, Tubingen, Germany. This study wasfunded in part by Merck KGaA. Merck KGaA did not influence the selec-tion of the patients, evaluation and acquisition of data, or the academicinterpretation of the data set.
tumors have many mutations in the isocitrate dehydrogenase1 and 2 genes (31), and IDH1 mutations are associated with abetter overall outcome in astrocytic neoplasms compared withwild-type tumors (32). Finally, methyl guanine methyltrans-ferase (MGMT) promoter methylation status has a prognosticrole and is an indicator of treatment response in GBM (33). Incombination with radiochemotherapy, the >vA3/>vA5 inhib-itor cilengitide is currently in a phase III trial (6) in GBM, themost malignant form of primary brain tumor.
Studies of integrin expression in human tumors havebeen limited by lack of antibodies capable of reacting withtargets in formalin-fixed, paraffin-embedded (FFPE) tissue, themethod routinely used in surgical pathology. Thus, data on ex-pression of target integrins >vA3 and >vA5 in brain tumorsis limited to a relatively few frozen tumor samples (15, 16, 20)and vital imaging studies (34). Moreover, longitudinal studiesare a profound logistic challenge. One of us (Simon Goodman)therefore developed a set of recombinant rabbit monoclonalantibodies (RabMabs) that 1) recognize integrins in FFPE ma-terial, 2) bind intact extracellular domains of the targets, and3) bind reproducibly using a standard automated immunohis-tochemistry (IHC) system (35). For this study, we used theseRabMabs against integrins >vA3, >vA5, >vA6, and >vA8 toanalyze their expression in a large longitudinal series of ar-chival FFPE normal brain, astrocytomas, and GBMs. Our dataindicate that expression of >vA3 in tumor cells and vessels,but not the other >v integrins, is dependent on tumor grade,and that its expression in tumor cells may have a prognosticimpact.
MATERIALS AND METHODS
AntibodiesMatched RabMabs directed against intact extracellular
domains of human >vA3, >vA5, >vA6, >vA8 complexes, thecommon >v subunit, and of the isolated A3-cytoplasmic do-main (cytoA3) were generated and characterized as described(35). Commercial antibodies to the integrins and ligands fi-bronectin and fibrinogen were also used (Table 1).
Tissue SamplesTumor and normal human brain samples were re-
trieved from the neuropathology archives of the Departmentof Pathology and Neuropathology, Institute of Pathology andNeuropathology, University of Tubingen, Tubingen, Germany(Table 2). Tissue handling was performed according to theethical guidelines of the University of Tubingen using a pro-tocol approved by the ethics committee (Permission No. 249/2010BO1). Histopathologic designation and grading of thetumors were done by at least 2 experienced neuropathologistsaccording to criteria of the 2007 World Health Organization(WHO) classification of tumors of the CNS (36).
Construction of Tissue MicroarraysOf the total of 503 tumors, 407 (81%) were available as
tissue microarrays (TMAs) (Table 2). Two 1,000-Km cylin-drical tissue core biopsies containing representative tumor tis-sue sections were punched from each paraffin donor block andtransferred into prepunched holes on recipient paraffin blocksat defined array coordinates. For each of the 9 autopsy cases
TABLE 1. Antibodies
Antibody to Clone, Species Dilution (concentration)Pretreatment, Primary Antibody
IgG IgG1 isotype control 1:500 (2 Kg/mL) Pretreatment and incubation times matchedto primary antibody
Genetex, San Antonio, TX
SCC1 (standard cell conditioner 1) and protease are reagents in the Benchmark Immunohistochemistry System (Ventana Medical Systems, Strasbourg, France).CytoA3, A3-integrin cytoplasmic domain.
TABLE 2. Tumor Samples and Number of Cases With Positively Stained Tumor Cells for Each Integrin Complex*
Tumor WHO Grade
No. Samples(No. Cases,
Female/Male)Mean age
(range), years >vA3 CytoA3 >vA5 >vA6 >vA8>vYCommon
*Data from manual assessment.†Ten samples from each autopsy case, including gray and white matter from frontal and occipital lobes, hippocampus, basal ganglia, cerebellum, mesencephalon, pons, brainstem.CytoA3, A3-cytoplasmic domain.
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diagnosed as normal human brain, a total of 10 differentregions (including gray and white matter of frontal and occipitallobes, hippocampus, basal ganglia, cerebellum, mesencephalon,pons, brainstem) were transferred to a recipient TMA block usinga TMA machine (Beecher Instruments, Inc., Sun Prairie, WI).After a short period of sealing at 37-C, the TMA blocks were cutwith a microtome (4-Km-thick sections) and mounted on Super-Frost Plus slides (Microm International, Walldorf, Germany).
ImmunohistochemistryImmunohistochemistry was performed on FFPE full-
slide tissue sections and microarrays after deparaffinization onan automated IHC system (BenchMark, Ventana Medical Sys-tems, Strasbourg, France), essentially as previously described(35). This system uses an indirect biotin-avidin system withstandard cell conditioner 1 and EDTA pretreatment protocol,a universal biotinylated immunoglobulin secondary antibody,and diaminobenzidine as chromogen. To improve signal qual-ity, the tissue sections were incubated with a copper enhancer(all reagents from iView DAB IHC Detection Kit; VentanaMedical Systems) and counterstained with hematoxylin. Pro-tocols were adapted to each primary antibody to achieve optimalsignal-to-noise ratio after initial antibody titration (Table 1).
Positive controls included normal kidney and malignantmelanoma for >vA3 and cytoA3, normal colon tissue and theHT-29 colon cancer cell line for >vA5, and normal kidney andthe HT-29 colon carcinoma cell line for >vA6 (35). Positivecontrols for >vA8 included normal human peripheral nerveand Ovcar-3 ovarian cancer cell line. For the antibody againstthe >v chain, which reacts with all of the >vAx complexes,normal colon tissue, HT-29 colon carcinoma cell line, andDU-145 prostate cancer cell line served as positive controls.Positive controls for fibronectin and fibrinogen included clear-cell renal carcinoma and GBM samples. Negative control slideswere processed parallel to each batch of staining by replacingthe primary antibody with the appropriate rabbit or murinepolyclonal immunoglobulin G (IgG) isotype control (Genetex,San Antonio, TX) at the same concentrations of IgG.
Mutation Analysis and Methylation-SpecificPolymerase Chain Reaction
DNA was extracted from GBM tissue sections from par-affin blocks (microscopically controlled for tumor content) usinga BlackPREP FFPE kit (Analytik, Jena, Germany), accordingto the manufacturer’s instructions. The IDH1 mutational statuswas assessed in WHO grade III and IV tumors by a mutation-specific monoclonal antibody for the IDH1 R132H mutationin tumor samples (32, 37). Negative cases (i.e. lacking theR132H mutation) were assessed by direct sequencing of therelevant exon for other rare IDH1 mutations (31). Bisulfitetreatment of DNA and analysis for MGMT promoter methyl-ation by methylation-specific polymerase chain reaction in WHOgrade IV tumors were performed as previously described (38).
Assessment of Clinical and Publicly AvailableGene Expression Data
Data on tumor localization, survival, age, sex, tumor type(primary or recurrent), edema, and Karnofsky score were re-trieved from the anonymized clinical files of all gliomas avail-able as TMA or full slides.
The NIH REMBRANDT database (Release 1.5.5, re-lease date: 07-27-2010, URL: https://caintegrator.nci.nih.gov/rembrandt/) was analyzed for possible effects of variations inITGB3, ITGB5, ITGB6, ITGB8, and ITGAV gene expressionon overall patient survival. REMBRANDT samples were di-vided into 3 groups based on mean expression levels of all re-porters (high, more than 2-fold; intermediate, 0.5- to 2.0-fold;and low, less than 0.5-fold) for median using the Li CancerRes2009 probe sets with Affymetrix chipsets (39). Survival analy-sis was performed directly with the database analysis tool (40).
Data Analysis and Statistical EvaluationStained TMA slides were scanned with a digital camera
(DFWX710; Sony, Japan) using a Mirax Scan system (Zeiss,Gottingen, Germany). Digitized data were transferred to aworkstation (Definiens Tissue Studio 2.0, Munich, Germany).After defining the tumor regions to be examined, stainingthresholds for nucleus detection and quantitative membraneand cytoplasmic intensity were defined on 4 subsets for subse-quent automatic analysis. Processed data (including number oftumor cells analyzed and their staining intensity) were then ex-ported into statistical analysis software.
In addition, the digitized stained slides were scoredmanually by 2 independent observers (Jens Schittenhelm,Esther Schwab). Expressions of integrin complexes and theirligands in vessels were semiquantitatively recorded as fol-lows: 0 (staining absent); 1 (staining in G50% of vessels); and2 (staining in Q50% vessels). Cytoplasmic and membranousparenchymal integrin expression intensity in tumor cells wasrecorded as follows: 0 (absent); 1+ (weak expression); 2+(moderate expression); and 3+ (strong expression).
Statistical analysis using JMP 7.0 (SAS Institute, Cary,NC) for automated analysis included analysis of variance ofthe score calculated for intensity and distribution (i.e. a his-toscore calculated as the percentage of tumor cells with weakexpression + double the percentage of tumor cells with mod-erate expression + triple the percentage of tumor cells withstrong expression; the system default setting provided by De-finiens Tissue Studio 2.0, Munich, Germany) followed by non-parametric testing (Wilcoxon for 2 pairs or Kruskal-Wallis for92 pairs). Logistic regression was used to compare staining datafrom the manual analysis with those from automated analysisand to compare IHC results with age. Correlation between tu-mor type, edema, sex, localization, IDH1 mutation status orMGMT promoter methylation status, and integrin complexscores was assessed using the Wilcoxon signed-rank test.
For the survival analysis, integrin subunit expression wassplit at the medians for the expression for each integrin com-plex in GBM, with subsequent Kaplan-Meier analysis. Signif-icances were determined using the logrank test. Survival datawere available for 130 patients (mean, 1.3 years; maximumfollow-up, 10.5 years).
RESULTS
>v Integrin Subunit ExpressionNumbers of positive tumor cases for each integrin com-
plex determined by manual evaluation of tumor cell distribu-tion are shown in Table 2. Integrin >vA3 was consistently
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expressed on cell membranes in endothelial cell proliferationswithin GBM (Fig. 1A). There was also >vA3 cytoplasmic/membranous staining in tumor cells that sometimes had a dis-tinctly perivascular pattern (Fig. 1B). Vascular >vA3 expres-sion was observed in endothelial cells and pericytes but wasabsent in perivascular smooth muscle cells and macrophages.Similarly, cytoA3 staining was observed in capillaries and inendothelial cell proliferations in GBM (Fig. 1C).
Cytoplasmic/membranous cytoA3 expression was pref-erentially found in large anaplastic GBM cells and perinecrotictumor regions (Fig. 1D). In approximately one fourth of thetumors, >vA5 expression was limited to vessels (Fig. 1E). Inthe remainder of the tumors, the distribution of cytoplasmic/membranous >vA5 was pronounced in perivascular tumor cells(Fig. 1F). Integrin >vA5 expression was always observed in en-dothelial cells, pericytes, perivascular monocytes/macrophages,and occasionally, in perivascular smooth muscle cells. Cyto-plasmic/membranous >vA8 was strongly expressed in mostglioma tumor cells; in many cases, only sparing the tumor ves-sels (Fig. 1G). Gliomas lacked >vA6 (Fig. 1H).
Cytoplasmic/membranous >v expression, although usu-ally strong, was absent in many tumors (Table 2). Ten regionsof 9 normal human brain autopsy cases were evaluated manu-ally for >vAx integrin expression (Table 2). No distinct >vA3expression was found in normal vessels or CNS parenchyma(Fig. 2A). There was homogeneous weak cytoA3 expressionin the basal ganglia in 2 cases (Fig. 2B), but expression wasabsent in other brain regions. Integrin >vA5 was seen in mostcapillaries of normal brain but not in the CNS parenchyma(Fig. 2C, arrows). As in gliomas, >vA6 was absent from nor-mal human brain (Fig. 2D). In some cases, there was prominentand homogeneous immunoreactivity for >vA8 in neocorticalneuropil, whereas adjacent vessels and white matter were un-stained (Fig. 2E). The >v complex was homogeneously ex-pressed in all human CNS regions and was also observed to aweaker extent in CNS vessels (Fig. 2F).
Integrin >v Complexes Staining IntensityScores and Tumor Grade
Integrin >vA3 expression intensity, as determined man-ually, was significantly higher in GBM than in anaplastic as-trocytomas, diffuse astrocytomas WHO grade II, and normalbrain (p G 0.0001) (Table 3A). The difference in >vA3 ex-pression between grade III and grade II neoplasms was notsignificant (Table 3A). Likewise, the mean intensity of ex-pression of the cytoplasmic A3 domain was significantly higherin GBM (p G 0.0001); expression was almost absent in astro-cytomas and normal brain, with no significant differences be-tween grades II and III and nontumor tissue.
Mean >vA5 expression intensity in GBM was signifi-cantly greater than that in anaplastic astrocytomas (p = 0.005)and diffuse astrocytomas WHO grade II (p = 0.01) (Table 3 B).The mean intensity in all tumors was significantly higher thanin normal brain (p G 0.0001), but the difference between gradeII and III tumors was not significant. The mean >vA8 ex-pression intensity was strongest in GBMs, with an expressionscore significantly higher (p G 0.0001) than expression levelsin anaplastic astrocytomas and WHO grade II diffuse astro-cytomas (Table 3 C). In all tumor groups, the mean >vA8
intensity scores were significantly higher than those in normalbrain (p G 0.0001); there were no significant intensity differ-ences between grade II and III tumors (Table 3C). Integrin>vA6 was absent from gliomas and normal brain (Table 3B).
As expected from the results of the staining intensity of>vAx complexes, mean >v expressions were strong in GBMsand anaplastic astrocytomas, with the highest levels in WHOgrade II diffuse astrocytomas (Table 3C). The differences inexpression between all 3 groups were highly significant (p G0.0001 to p = 0.0032). The expression of the integrin ligandsmatched the pattern seen in the >vAx complexes. Whereasthere was a significant and progressive increase of mean fibro-nectin intensity from grade II diffuse astrocytomas (p = 0.001,II vs III; p G 0.0001, II vs IV) over anaplastic astrocytomas toGBM (p G 0.0028 for III vs IV), the mean expression inten-sity of fibrinogen was significantly lower in grade II and IIItumors (no statistical differences between tumor grades II andIII) than in GBM (p G 0.0001) (Table 3D).
Association Between Staining Intensitiesof >v Integrin Complexes and Ligands
Multivariate analysis of all tumors followed by pairwisecorrelations of manual staining intensity revealed a significantpositive association between integrin complexes >vA3, >vA5,>vA8 and cytoA3 (p G 0.0001 to p = 0.0002). There was asignificant positive correlation with the >v subunit for >vA5(p = 0.0306) and cytoA3 (p = 0.0009) but not for >vA3 (p =0.441) and >vA8 (p = 0.729). The positive association of theA complexes with the staining intensity of the ligands fibrin-ogen (p G 0.0001) and fibronectin (p = 0.0020) was alsohighly significant.
Integrin >vA3 and >vA8 Staining and OtherTumor Features
The mean >vA3 (p = 0.084), A3 cytoplasmic domain (p =0.0053), >vA8 (p = 0.0002), >v (p G 0.0001), and fibronectin(p = 0.0002) staining intensities were less in high-grade tumorscarrying the prognostic IDH1 R132H mutation (Table 3A,C, D); the mean >vA5 intensity was similar in both groups(Table 3B). There was no difference in >v integrin expressionintensity between GBMs expressing methylated or unmethylatedMGMT promoters (Table 3AYD). The intensity of staining ofintegrin complexes in relapsing tumors was the same as that inprimary neoplasms (Table 3AYD). The mean intensity sig-nificantly increased with patient age for >vA3 (p = 0.0007),cytoplasmic A3 (p = 0.0002), >vA8 (p G 0.0001), fibrinogen(p G 0.0001), and fibronectin (p = 0.00017), whereas >vA5and >v expression levels were not significantly different be-tween age groups (Table 3AYD). These results did not retainsignificance after the tumors were separated according to theirWHO grade and age was correlated with mean integrin ex-pression intensity levels in these subgroups; however, therewas an age-dependent increase in expression of >vA8 (p =0.0249) and fibrinogen (p = 0.003) in GBM (data not shown).
Patients with lower Karnofsky scores exhibited slightlyhigher mean >v integrin intensity than those with higher scores(Table 3AYC), except for >vA5. After separating tumors ac-cording to their WHO grade, the results were no longer sig-nificant, except for >vA8 in diffuse astrocytoma (p = 0.0273)
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FIGURE 1. Immunohistochemical expression patterns of integrin complexes in glioblastoma samples. (A, B) Expression of >vA3 intumor vessels ([A] arrows) and with additional parenchymal >vA3 immunoreactivity ([B] asterisks). (C, D) Expression of A3cytoplasmic domain (cytoA3) in vessels is stronger than in the surrounding tissue (C) and is similar to that in perinecrotic areas (D).Tumors with >vA5 expression restricted to tumor vessels (E) and with additional parenchymal >vA5 immunoreactivity (F). (G)Expression of >vA8 is absent in vessels (arrows) but usually is strong in tumor parenchyma. (H) Integrin >vA6 is absent in gliomacells and vessels. Scale bar = 100 Km.
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FIGURE 2. Immunohistochemical expression patterns of integrin complexes in normal human brain samples. (A) Lack of >vA3expression in basal ganglia (inset: melanoma cell line as a positive control). (B) Weak and diffuse A3 cytoplasmic domain (cytoA3)expression in basal ganglia. (C) Expression of >vA5 in basal ganglia is restricted to microvessels (arrows). (D) Integrin >vA6 isabsent in normal brain (inset: adenocarcinoma of the intestine as a positive control). (E) Neocortical >vA8 expression is slightlyenhanced in subpial neuropil regions but is absent in vessels. (F) Neocortical >v complex expression in neuropil and in vessels(arrow). Scale bar = 100 Km.
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and fibronectin (p = 0.0268) in GBM (Table 3D). No signif-icant associations were found between sex, tumor localiza-tion, or edema and integrin intensity.
Vascular >vA3 and Cytoplasmic A3 ExpressionsIncrease With Tumor Grade
Vessel staining was analyzed only in the 63 tumor sam-ples available as full slides because not all TMA punches con-tained scorable vasculature. Integrins >vA6 and >vA8 wereabsent in tumor vessels (Fig. 1G, H). There was significantupregulation of >vA3 (p G 0.0001) and cytoplasmic A3 (p G0.0001) distribution in tumor vessels that correlated with thegrade of malignancy, whereas the distribution of other inte-grin complexes remained stable (i.e. no significant differ-ences among tumor grades). In addition, the expression of theligands fibronectin (p G 0.0001) and fibrinogen (p = 0.0007) inGBM vessels showed a significant increase with tumor grade(Fig. 3D).
Correlations of Digital and Manual StainingThe logistic fit of manual intensity scoring with the
histoscores calculated by automated intensity and distributionanalysis with the Definiens image processing software wasanalyzed where available (n = 55; only TMA). The 2 methodsshowed a significant association for >vA3 (p G 0.0001), cytoA3(p = 0.0005), >vA5 (p G 0.0001), >vA8 (p = 0.0001), >v (p G0.0001), fibrinogen (p G 0.0001), and fibronectin (p = 0.0009)expression. Integrin >vA6 was not expressed.
Staining Intensities and Tumor GradeAfter the overall concordance between automatic and
manual data was confirmed, 386 brain tumor TMA cores wereevaluated by detailed automated cell-based analysis for thestaining intensity and distribution of each integrin complex(Fig. 3). Each TMA core was segmented into cell fields de-pending on the nuclei present in the immunostained specimen.An average of 3,853 fields on a single TMA core with a meandiameter of 70.3 Km for each field were available for a detailedanalysis of staining intensity (Fig. 3A). Mean percentages oftumor cell segments showing absence or weak, moderate, andstrong expression in all fields were calculated (Fig. 3B).
The digitized >vA3 microarray samples of high-gradegliomas (WHO III and WHO IV) showed a higher mean per-centage of tumor cells exhibiting moderate (17% in WHO III;13% in WHO IV) and strong staining intensity (20% in WHOIII; 14% in WHO IV) than in grade II tumors (p = 0.037, 11%moderate; p = 0.013, 12% strong), whereas the frequency oflow expression intensities (68% in WHO II; 58% in WHO III;69% in WHO IV) was similar. This analysis included some
capillary endothelial cells that could not always be excluded;however, the mean percentage of tumor cells negative for>vA3 was higher in GBM (22%) than that in grade II (7%)and III neoplasms (4%) (p G 0.0001; Fig. 3B), indicating that asubset of GBM loses >vA3 during tumorigenesis/progression.Analysis of >vA5 showed that the distribution of staining in-tensity was unchanged between tumor grades.
In GBM, the mean percentage of >vA8 high-expressingcells was significantly higher (44%; p G 0.001) than that ingrade II (8%) and grade III (8%) tumors, indicating an increasein intensity accompanying tumor progression. By contrast, fewerGBM showed a low mean >vA8 staining intensity (to 28%, p G0.001; Fig. 3B) than grade II (71%) and grade III (67%) tumors.There was also a slightly increased mean percentage of tu-mor cells with moderate >vA8 immunoreactivity in GBM(26%) compared with those in astrocytoma grade II (17%)and grade III (16%).
Analysis of the >v integrin subunit showed that thenumbers of >v high-expressing cells were lower in grade II(17%) and III (9%) tumors than in grade IV tumors (51%, p G0.0001), whereas >v low-expressing and immunonegative cellswere more frequent in grade II (low, 19%; absent, 36%) andIII tumors (low, 10%; absent, 68%) than in GBMs (low, 8%;absent, 9%; p = 0.0013 to p G 0.0001), independently indi-cating that there was a general increase in >v integrin ex-pression during tumor progression.
Combined Intensity and Distribution HistoscoreGenerated by Automated Analysis
The intensity and distribution results of the automatedanalysis (Fig. 3B) were combined into a histoscore for eachcomplex and tumor grade (Fig. 4). The significance of themean histoscores was divergent for >vA3, possibly repre-senting an activation epitope (p = 0.02 between grades II andIII; nonsignificant between grades III and IV), and cytoplas-mic A3 (nonsignificant between grades II and III; p = 0.0039between grades III and IV), representing the overall protein(35). In contrast, the mean histoscores for >vA5 did not differsignificantly between the tumor grades, and, as expected, >vA6was completely absent. The mean >vA8 and >v histoscoreswere significantly higher in GBM (p G 0.0001) than those ingrade II and III tumors (Fig. 4).
ITGB3 and ITGB5 Gene Expression in anIndependent Data Set
We attempted to validate the results of the IHC analy-sis independently at the level of gene expression. We ana-lyzed the gene expression of the relevant integrin chains
Present 31 0.12 0.42 0.0002 31 0.35 0.35 0.24
MGMT promoter methylation
Methylated 9 1.11 1.16 10 0.50 0.52
Unmethylated 11 1.25 0.62 0.75 12 0.91 1.08 0.25
For each antibody, the number of tumors, mean intensity scores, and SD are shown. Results of the multivariate analysis are from Wilcoxon testing if not otherwise specified.*Kruskal-Wallis test.†Logistic fit.‡High-grade WHO III and IV gliomas.IDH1, isocitrate dehydrogenase 1; MGMT, methyl guanine methyltransferase.
TABLE 3. (Continued)
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FIGURE 3. (A) Immunohistochemical staining for >vA5 of a glioblastoma tissue microarray sample (left) and an example of adetailed computed analysis after segmentation of the same sample into cell-based fields (right). The colors indicate quantificationof expression within each field: (white, no staining score; yellow, weak staining, 1+; orange, moderate staining, 2+; Bordeaux redmagenta, strong staining, 3+). (B)Mean percentage of cells scored as 0 to 3+ of all tissue microarrays stratified for tumor grade andanalyzed for each integrin complex. >v-, pan->v integrin; cytoA3, A3 integrin cytoplasmic domain.
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in the Unified Gene data set NIH REMBRANDT (40). Thisanalysis revealed that ITGB3 expression increased during pro-gression from normal human brain (median, 39.3) to astrocy-toma tissue (median, 59.0), with the highest levels in GBM(median, 100.3). Expression of ITGB5 in tumors (medianGBM, 1,048; median astrocytoma, 853.5) was also higher than
that in normal brain (median, 532). Expression of ITGB8 geneincreased during the progression from nontumor tissue (median,167.4) to astrocytoma (median, 294.7) and then to GBM (me-dian, 351.1). The ITGB6 gene expression intensity in astrocy-tomas (median, 26.7) and GBMs (median, 23.8) did not exceedbaseline levels found in normal brain (median, 34.1). Thus,
FIGURE 4. Combined histoscore results of an automated analysis of staining intensity and distribution of immunoreactive paren-chymal tumor cells for each integrin complex differentiated according to WHO tumor grade. The number of tumor samples isshown for each grade. Means are depicted by bars.
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the gene expression data from an independent sample ofnormal and tumor patients closely correlated with the protein-based longitudinal IHC data from our brain tumor FFPE archive.
Survival AnalysisStatistical analysis of the survival plots of patients from
the current IHC study split at the median histoscore from theautomated analysis of GBM (Fig. 4) showed a significantlybetter overall survival for those with no or low expression ofthe >vA3 complex (logrank, p = 0.016; median histoscore usedfor splitting, 67; number of samples analyzed, 39) than forthose with high expression (i.e. above the median). In con-trast, no significant association was seen between patient sur-vival and integrin >vA5 (logrank, p = 0.46; median, 82; n =47), A3 cytoplasmic domain (logrank, p = 0.07; median, 49; n =57), >v (logrank, p = 0.2225; median, 155; n = 30), or >vA8(logrank, p = 0.93; median, 211; n = 58) expression in GBM(Fig. 5).
Correlation of ITGB3 gene expression from the NIHREMBRANDT database and patient survival revealed a signif-icantly poorer outcome for those showing high ITGB3 levels(i.e. 92-fold gene expression changes; patients, n = 53) thanfor those with intermediate and low ITGB3 transcript levels(G2-fold expression change; n = 203; logrank, p = 0.0022)(Fig. 6). Patients with more than 2-fold ITGB5 upregulation (n= 77) showed a trend toward poorer survival compared withthe ITGB5 intermediate and low groups (n = 177; logrank, p =0.06). No significant differences were observed for the ITGB6upregulated and intermediate/low ITGB6Yexpressing cohort(p = 0.98). By contrast, significantly poorer survival was notedin the cohort with high ITGB8 transcript levels (n = 188)compared with those with intermediate and low ITGB8 tran-script levels (n = 68; logrank, p = 0.0006). Likewise, the cohortwith high ITGAV transcript levels (n = 94) showed signifi-cantly poorer survival than the intermediate/low ITGAV tran-script group.
DISCUSSIONSeveral agents can modulate cell attachment, differen-
tiation, and migration through inhibition of >vA3 and >vA5integrins (1, 23, 41, 42), which are current therapeutic targetsin human gliomas (42, 43). In this study, we examined theexpression of >v integrins by IHC in archival FFPE braintumor specimens and compared this with historical gene ex-pression data in a different patient population recorded in theNIH REMBRANDT expression database (39). Our principalfindings are 1) that expressions of >vA3 and >vA8 protein andITGB3 and ITGB8 mRNA increase during glioma progres-sion; 2) that patients with above median protein expression atdiagnosis for >vA3 have a poorer prognosis than those withbelow the median, but this does not hold for >vA5 or >vA8; 3)that the same poorer prognosis for ITGB3 overexpression isseen in the NIH REMBRANDT sample (in addition to a poorprognosis associated with ITGB8); and 4) that automated sam-ple processing and data collection by IHC using a novel set ofRabMabs against >v integrins can provide a robust prognos-tic signal in glioma patients. This cross validation of proteinand mRNA expression data strongly indicates that >v inte-grins, and especially >vA3, are independent prognostic indi-
cators in gliomas. An >vA3/>vA5 inhibitor, cilengitide, iscurrently in a phase III clinical trial for glioma (6, 42, 44), andit will be interesting to compare the outcome of this trial withthe >v integrin expression profiles of the patients.
To date, analysis of integrins in human pathology spec-imens has been hampered because most of the available anti-bodies stain only frozen tissue, which is rarely accessible forlongitudinal study, whereas antibodies detecting integrins inFFPE specimens are generally lacking. Thus, the type of studyof integrin profile in archival materials described here wasessentially not possible in the past. Newly developed RabMabsagainst integrin >v complexes (35) show easily interpretablestaining patterns in FFPE tissue, including samples of nor-mal brain, diffuse astrocytomas, and GBMs. They can be usedon standard IHC autostainers using standardized validatedprotocols.
In this study, there was excellent correlation betweenthe scores performed independently by a pathologist and anautomated computer analysis for staining intensity and dis-tribution. This validates the analysis algorithm used (45) andindicates that the methods are suitable for evaluating largenumbers of specimens. The automated method also provideddetailed data for each field, approximately representing ananalysis of individual tumor cells. This allowed us to detectshifts of expression within tumor cohorts that are often hid-den within an overall staining score, for example, what isroutinely applied in immunopathology to regions of hetero-geneous expression. A pathologist must still instruct the soft-ware during automated analysis about which regions of thesection are to be analyzed because the analyzer cannot dis-criminate between normal, necrotic, or neoplastic tissue anddoes not fully exclude smaller vessels. The current drawbackis the time-consuming step of digitizing individual slides andselecting regions for analysis. We have found that construc-tion of TMAs is the method of choice to optimize the auto-mated analysis and save valuable time.
The integrin >vA3 is a promiscuous receptor for com-ponents of the provisional ECM, including vitronectin, fibrin-ogen, and fibronectin, and is found in normal smooth musclecells, osteoclasts, monocytes, and platelets (46). Integrin >vA3is upregulated in gliomas, melanomas, and carcinomas (2, 11,19), and its expression is deregulated in autoimmune diseasesand transplant rejection (6, 12). In glioma cell lines, upregu-lation of >vA3 can increase their sensitivity to anticancer ra-diotherapy and to drug-induced apoptosis (14, 47). Endothelial>vA3 enhances the activity of vascular endothelial growthfactor and platelet-derived growth factor, which are frequentlyexpressed in malignant gliomas (48, 49). Using relatively smallnumbers of tumor frozen sections, Gladson (16) and Bello et al(15) demonstrated that >vA3 expression occurs in glioma endo-thelial cells. We were now able to confirm these observationson a large number of FFPE tumors, that is, >vA3 is indeed ex-pressed in glioma tumor vessels and is upregulated with gradeof malignancy, further supporting a role of >vA3 for vasculardevelopment in GBM. Using the monoclonal antibody LM609,Bello et al (15) reported >vA3 to be highly expressed in 35%of tumors (14 cases), moderately in 18% (7 cases), and weaklyor sporadically in 30% (12 cases). In our >vA3 analysis ofFFPE specimens, however, we obtained results more similar
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FIGURE 5. Kaplan-Meier survival analysis of patients expressing >vA3 (upper left, low [red]/high [blue], n = 17/22), A3 cytoplasmicdomain (cytoA3) (upper right, low/high, n = 35/22), >vA5 (middle left, low/high, n = 14/33), >vA8 (middle right, low/high, n = 26/32), and >v (lower left, low/high, n = 12/18) assessed by immunohistochemistry using rabbit monoclonal antibodies in glioblas-tomas split at the median histoscore. Integrin >vA6 was excluded because no positively stained tumors were available (scale barsare tumor dependent because not all tissue microarray tumor punches were available for all integrin complex stains).
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to a later study (20): 53% of our GBM expressed >vA3-immunopositive cells. Furthermore, we determined that pa-renchymal expression of >vA3, although present even in some
low-grade tumors, is indeed correlated with increased tumorgrade both in immunostains and gene expression analyses andis also associated with a poorer prognosis compared with
FIGURE 6. Kaplan-Meier survival analysis of glioma samples from an NIH REMBRANDT data set (x axis: days of observation) withmean upregulated ITGB3 gene expression (blue) compared with those with intermediate (red) and low ITGB3 expression. Logrankp values are shown for each gene inside the graph.
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>vA3 parenchymalYnegative tumors. There was no such cor-relation at the >vA5 and >vA8 protein level. Moreover, ex-pression of >vA3 was reduced in IDH1-mutated gliomas,probably further affecting the prognostic differences betweenthese groups (32, 37). This is important because integrin >vA3and >vA5 inhibitors affect both tumor vessels and parenchy-mal cells (16, 21, 24). In contrast to >vA3, expression of >vA5in GBM vessels and parenchymal cells remained constant inour series, indicating a major role for >vA3 in gliomas. Sur-prisingly, we did not find an association between any of theintegrin complexes and MGMT promoter methylation, asGBM patients carrying the MGMT methylation have a betterprognosis under combined temozolomide, radiotherapy, andcilengitide treatment (50). Further studies on the possible prog-nostic relationship of MGMT and >vA3 are needed.
In principle, the antibody for the cytoplasmic domainof A3 (EM002-12) and the anti->vA3 antibody (EM227-03)should have similar tissue staining patterns outside the vas-culature. Whereas the intensity results were similar (Table 3A),the combined intensity and distribution scores were divergent(Fig. 4). The >vA3 antibody (EM227-03) can recognize anepitope expressed more strongly on ligated integrins, whereasthe cytoplasmic A3 reagent (EM002-12) should see all avail-able >vA3. Thus, the difference between the poorer prognosisof >vA3 (i.e. EM22703 high expressers) and the significantlybetter prognosis for low expressers and the similar survival ofthe high- and low-expressing cytoA3 population is of inter-est. Perhaps, ligated >vA3 integrin in patients with poor prog-nosis is related to their outcome, which would justify a targeted>vA3 therapy (43). However, it is unclear whether EM22703reacts with activated >vA3 in situ, so this remains a specula-tion. Publicly available data on integrin gene expression fromthe REMBRANDT files also reveal a poor outcome for gliomapatients in whom ITGB3 was upregulated, which substantiatesour data on patients with high >vA3 protein expression as as-sessed by IHC.
Integrin >vA5, a vitronectin receptor, has been reportedin several types of cancer and established cell lines (35, 51Y53).In carcinomas, interaction between EGFR and integrin >vA5affects metastatic and invasive cell potential (54). Recent dataindicate that there is parenchymal >vA5 expression in gliomasand in angiogenic endothelial cells (27). Previous studies in-dicate that the number of integrin-positive cells in frozenglioma tissue is between 30% and 85%, depending on the anti-bodies and evaluation methods used (15, 20, 55). The observeddistribution heterogeneity in tumor samples may result fromhypoxia-dependent induction of >vA5 in gliomas (56). Over-all, our data indicate that the role of >vA5 in glioma is minorcompared with that of >vA3.
The epithelial fibronectin receptor >vA6 is upregulatedduring wound healing and carcinoma progression and can mod-ulate matrix metalloproteinases and transforming growth factor-A1 activation (7). Expression has been previously reported ingastrointestinal, pancreatic, and esophagus adenocarcinomas(9). Upregulation of >vA6 in nonYsmall-cell lung carcinomacorrelates with shorter survival (57). There was no >vA6 im-munoreactivity in our glioma series or in normal brain, al-though the EM05201 antibody strongly stained controls (35)(Fig. 2D), which we confirmed on more than 100 carcinoma
samples (data not shown). Because integrin >vA8 was ex-pressed in most gliomas (~95%), regardless of WHO stage, acombined >vA8-positive/>vA6-negative immunoprofile mighthelp determine whether a tumor is of glial originVwhen theglial fibrillary acidic protein immunophenotype in late-stagededifferentiated GBM is equivocal (58). Integrin >vA6 hasnot been reported in the brain, and it is classically an epithe-lial integrin that is upregulated in carcinoma (59). Therefore,our confirmation of its absence in glioma and normal brain isnot surprising, but the brain phenotype of >vA8 comparedwith the epithelial phenotype of >vA6 is notable particularlybecause both can assist activation of transforming growthfactor-A1 (60).
Expression of >vA8 integrin has been reported for hu-man glioma cell lines (61), in neuronal progenitor cells (61),and in neuronal and glial cells in rodent brain (62, 63). InEGFR/PDGFRA-overexpressing gliomas, >vA8 is upregulated,often in a perinecrotic or perivascular pattern (64). Paren-chymal >vA8 plays a role in vascular development and in thepathogenesis of brain arteriovenous malformations (65, 66).Here, automated analysis of >vA8 showed a transition fromweak to strong expression in tumor cells with increasing gradeof malignancy. These tumor cells were often perivascular. Wefurther corroborated the reported high expression of A8 mRNAin primary GBM (64) using the REMBRANDT gene data set.Because, in the postnatal brain, >vA8 integrin has been re-ported in neural stem cells and in progenitor cells (67), theconsistent A8 expression in glioma was possibly caused byreactivation of a glioneuronal phenotype. The lack of >vA8in human tumor vessels parallels the lack of >vA8 in rodents(63). In none of these studies were the reagents capable ofdetecting intact >vA8 complexes, however. This complicatesthe interpretation of the data sets, unlike the unequivocal stain-ing of our >vA8 antibody (EM13309), which binds the intactextracellular domains of the integrin >vA8 heterodimer. Thereis evidence that the tumor vascular pathology and hemorrhageseen in high-grade gliomas may be related to loss of paren-chymal >vA8 (61, 63, 65). Inasmuch as we observed in-creased >vA8 expression in tumor cells in GBM, >vA8 eitheroverexpression or underexpression may affect vessel forma-tion. Our observations of >vA8 expressed in tumors derivedfrom astrocytes apparently conflict with some cellular data onthe expression of >vA8 integrin. Using the antibodies usedhere, Tchaicha et al and one of us found little >vA8 on U87glioma (35, 60, 61). Some GBM lines clearly can express >vA8,which may reflect their invasive capability (61). It has alsobeen reported that >vA8 in U87 glioma can be suppressed bymiRNA93 transfection (68). Because expression of miRNA93is also significantly different in GBM compared with U87MGcells (69), this might also affect >vA8 expression in the cellline. Furthermore, cell lines can switch their protein expressionprofile during adaptation to culture conditions, whereas IHCreveals the primary status of the tumor in situ. Provided thatthe antibodies are well characterized and the staining condi-tions are well validated, IHC data on expression in situ shouldtake precedence over data derived from cell lines in vitro. Inaddition, the models used by Tchaicha et al (61) lack the typ-ical pseudopalisading necrosis and glial fibrillary acidic pro-tein expression of human GBM and were genetically more
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closely related to secondary GBM. This may indicate differentroles for >vA8 in different tumor subpopulations that needfurther study. Finally, >vA8 data from cell lines need to be in-terpreted cautiously because the origin of several ‘‘glial’’ lineshas now been challenged (70).
In conclusion, we used newly developed rabbit mono-clonal antibodies for fast automated histopathologic determi-nation of >v integrin complexes in long-term archival gliomaspecimens. Overexpression of >vA3 integrin overtly signifiesa poor prognosis in glioma and might help in stratifyingpatients for anti-integrin treatment by IHC, whereas otherintegrins of the >v series do not. Finally, the detailed semi-automated quantification of parenchymal >v expression inparaffin blocks will allow us to easily correlate integrin ex-pression data from individual patients with their therapeuticresponse to integrin inhibitor treatment.
ACKNOWLEDGMENTSThe authors thank Katrin Trautmann and Karen Petersen
for help with additional immunostaining. Research antibodiesEM227-03, EM002-12, EM099-02, EM052-01, EM133-09,and EM013-09 were kindly provided by Merck KGaA,Darmstadt, Germany.
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