vascular patterns in glioblastoma influence clinical outcome and associate with variable expression...

Peter Birner MD 1,2; Maria Piribauer MD 3; IngeborgFischer MD 2; Brigitte Gatterbauer MD 4; ChristineMarosi MD 3; Peter F. Ambros PhD 5; Inge M. Ambros,MD5; Markus Bredel MD 2*; Georg Oberhuber MD 1;Karl Rössler MD 4; Herbert Budka MD 2; Adrian L. Har-ris DPhil 6; Johannes A. Hainfellner MD 2

1 Institute of Clinical Pathology, 2Institute of Neurology, 3Depart-ment of Internal Medicine I, 4Department of Neurosurgery,University of Vienna, Austria.

5 Children’s Cancer Research Institute (CCRI), St. Anna ChildrenHospital, Vienna, Austria.

6 ICRF Molecular Oncology Laboratory and AngiogenesisGroup, Institute of Molecular Medicine, John Radcliffe Hospi-tal, Oxford, United Kingdom.

* Current address: Department of General Neurosurgery, Neu-rocenter, University of Freiburg, Freiburg, Germany

No data exist on angiogenic patterns and theirprognostic impact in human glioblastoma. Suchdata are relevant for translation of antiangiogenictherapies into clinical applications. Using immuno-histochemistry for CD34, we assessed vascular pat-terns in 114 primary glioblastomas. Vascular pat-terns comprised unevenly distributed glomeruloid/garland-like/clustered bizarre vascular formationsand evenly distributed delicate capillary-likemicrovessels (“classic” vascular pattern). The com-bination of low content of bizarre vascular forma-tions and prominent classic vascular pattern (n = 29)was an independent factor for longer survival (p =0.006, Cox regression), as well as postoperativehigh Karnofsky performance status (p = 0.005). Inpatients with a prominent classic vascular pattern,there was no difference of MIB1 labeling indexwhereas microvessel density and apoptotic index(TUNEL) were significantly higher as compared to allother patients (p<0.05). In addition, diffuse expressionof hypoxia-inducible factor (HIF)-1 � and strongexpression of vascular endothelial growth factorwere more common (p<0.05, Chi-square test). FISHrevealed loss of chromosomes 1p and 19q only in 1/7long-time survivors with classic pattern. We con-clude that vascular patterns in primary glioblastomainfluence clinical outcome and associate with variableexpression of angiogenic proteins. Our findingsdenote for the first time distinct angiogenic sub-

types of human glioblastoma which may prove rele-vant for anti-angiogenic therapy approaches.

Brain Pathol 2003;13:133-143.

IntroductionIrrespective of all treatment efforts median survivaltime in glioblastoma remains below 12 months (18). Asconventional therapies, ie, operation and adjuvantradio-/chemotherapy, fail to prolong survival for morethan a few months, brain tumor research has beenfocused in recent years on novel therapy approachesbased on molecular insights in brain-tumorigenesis. Animportant field of research is the pathophysiology ofangiogenesis and development of antiangiogenic thera-pies for glioblastoma (12, 23, 37). Angiogenesis andcellular adaptation to hypoxia represent a key step intumor progression in general (25, 48). In glioblastoma,unevenly distributed bizarre vascular formations, ie,glomeruloid vascular formations, vascular garlands andvascular clusters, are commonly observed, in addition to“classic” angiogenesis, evident by evenly distributedcapillary-like microvascular sprouting (17, 31, 42).These bizarre vascular formations are a histologicalhallmark of glioblastoma (17) and are considered as a late,secondary event that is insufficient to save the endangeredtumor tissue from hypoxia-mediated death (31, 42). It hasbeen suggested that the efficacy of antiangiogenic ther-apy for glioblastomas may be determined by the degreeof classic angiogenesis with the formation of delicatemicrovascular sprouts (42). However, no data exist onangiogenic patterns and their prognostic impact. Suchdata are relevant for translation of antiangiogenic ther-apy from the experimental stage into clinical application.In our study we show for the first time that vascularpatterns in glioblastoma influence clinical outcome andassociate with variable expression of angiogenic proteins.

Materials and Methods

Patients. All patients who underwent initial surgeryof primary glioblastoma between January 1995 and

RESEARCH ARTICLE

Vascular Patterns in Glioblastoma InfluenceClinical Outcome and Associate with VariableExpression of Angiogenic Proteins: Evidence forDistinct Angiogenic Subtypes

Corresponding author:Johannes A. Hainfellner, Institute of Neurology, AKH 4J, POB 48, Währinger Gürtel 18-20, A-1097 Vienna, Austria(e-mail: [email protected])

December 1999 at the University Hospital of Viennawere included in this retrospective study. Recurrenttumors or tumors with a history of a previous low-gradeastrocytoma were not used in this study. All tumor spec-imens were reviewed and confirmed as glioblastomaaccording to the current WHO definition (17). None ofthe tumors displayed oligodendroglial features.

Extent of surgery was documented by the neurosur-geons as subtotal or total resection.

Postoperative Karnofsky performance status wasdetermined 10 to 14 days after surgery (16).

Radiotherapy was administered to a total dose of 66Gy (2 Gy/fraction) or 51 Gy (3 Gy/fraction) within 5 to6 weeks after surgery. Nitrosurea-based chemotherapy wasstarted 10 to 14 days after surgery. In patients older than50 years and Karnofsky performance status ≥60, 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea (CCNU) wasadministered orally at 100 mg/m2 in 6 to 8 week intervalsor 1-(4-amino-2-methyl-5-pyrimidinyl)-methyl-3-(2-chloroethyl)-3-nitrosourea (ACNU) intravenously at100 mg/m2 in 6 weeks interval. Patients younger than 50years and Karnofsky performance status ≥60 received acombination of dacarbacine (200 mg/m2) and fotemustine(100 mg/m2) every 3 weeks intravenously.

Methods. Expression of CD34, Ki-67, HIF-1�, HIF-2�, p53, epidermal growth factor receptor (EGFR) andvascular endothelial growth factor (VEGF) proteins andapoptotic index were determined in paraffin-embeddedtumor specimens fixed in 4% buffered formalin. Histo-logical slides, 4 �m in thickness, were deparaffinized inxylol. For anti-CD34 staining a monoclonal antibody wasused (antibody QBEnd/10, BioGenex, San Ramon,Calif) according to a standard protocol (32). Immunos-taining for KI-67 and p53 proteins was performed usingthe monoclonal antibodies MIB1 and DO-7, respec-tively (both from DAKO, Glostrup, Denmark), accord-ing to standard protocols (5). Apoptotic index of tumorcells was determined using in situ-end labeling with theTUNEL assay (Roche Diagnostics, Mannheim, Ger-many) according to the manufacturer’s instructions. Forimmunohistochemical detection of HIF-1�, EGFR andVEGF, slides were heated in 0.01 M citrate buffer (pH6.0) for 30 minutes in a microwave oven at 600W. Aftercooling for 20 minutes and washing in PBS, endogenousperoxidase was blocked with methanol containing 0.3%hydrogen peroxide for 30 minutes, followed by incuba-tion with PBS containing 10% normal goat serum for 30minutes. Specimens were incubated overnight at +4°Cwith a monoclonal anti HIF-1� antibody (BD Trans-duction Laboratories, Franklin Lakes, NJ, #H72320)

(44) at a dilution of 1:25 or a polyclonal rabbit antibodyagainst VEGF (Santa Cruz Biotechnology, Santa Cruz,Calif, #sc-152) (19) at a dilution of 1:100. EGFRexpression was determined using a monoclonal anti-body (NCL-EGFR; Novocastra Laboratories, Newcas-tle, United Kingdom) at a dilution of 1:20 with an incu-bation time of 1 hour. Immunostaining of HIF-2a wasperformed using the monoclonal antibody EP190b, asdescribed previously (39). Detection of immunostainingwas performed using the ChemMate kit (DAKO,Glostrup, Denmark) and diaminobenzidine or 3-amino-9-ethylcarbazole (for VEGF- immunostaining) as chro-mogene. As positive control for HIF-1� expressionimmunostaining was also performed on 2 samples ofovarian cancer with known strong expression of HIF-1�,which have also been used in a previous study (6). As pos-itive controls for VEGF, CD 34, p53, and HIF-2�,appropriate specimens of breast cancer were used. Twosamples of squamous cell cancer served as positivecontrols at EGFR- immunostaining. For negative control,primary antibodies were replaced by non-immune iso-typic antibodies or normal rabbit serum, as appropriate.Triple-labeling immunofluorescene was performedusing the CD34 antibody and antibodies againstCD105/endoglin and Id-1 (33) (both from Santa CruzBiotechnology, Santa Cruz, Calif). For detection ofantibody binding, Alexa Fluor 488, 546 and 633 labeledisotype-specfic secondary antibodies (MolecularProbes Inc., Eugene, Ore) were used according to themanufacturer´s instructions. Confocal laser scanningmicroscopy was performed using a Zeiss LSM 510(Zeiss, Oberkochen, Germany).

Fluorescent in situ hybridization (FISH) for loss ofchromosomes 1p and 19q was performed on sediment-ed tumor cell nuclei isolated from the formalin-fixed,paraffin embedded material. 1p analysis was performedwith the paracentromeric probe D1Z1 (1q12) (9) and sub-telomeric probe D1Z2 (1p36.3) (Q-BIOgene, Heidel-berg). For 19q analysis, probes 19pter and 19q13.3were used (both Vysis, Bergish-Gladbach, Germany).Assessment and interpretation of FISH results was doneaccording to guidelines defined by the InternationalSociety of Pediatric Oncology (E-SIOP NeuroblastomaStudy Group) for studies of peripheral neuroblastictumors (1).

Analysis. Analysis was performed by threeobservers (P.B., I.F., J.A.H.) blinded to clinical datausing a multiheaded microscope.

Analyzing anti-CD34 immunostained sections, weclassified vascular patterns as follows. Bizarre vascular

134 Vascular Patterns in Glioblastoma: Evidence for Distinct Angiogenic Subtypes—Birner et al

formations: 1. glomeruloid vascular proliferations:clustered vessels ensheathed by connective stroma (17)(Figure 1A); 2. vascular garlands: clustered vesselswith and without connective stroma arranged in gar-land-like formations, frequently around necroses (Figure1B); and 3. vascular clusters: distinct focal aggrega-tions of vessels (≥3) without connective stroma (Figure1C). Microvascular sprouting: delicate capillary-likemicrovessels resembling classic angiogenesis, distributed

evenly throughout major parts of vital tumor tissue(Figure 1D).

To assess the predominance of vascular formationssemiquantitatively, we defined a scoring system as fol-lows: Glomeruloid vascular proliferations: 0 points: noglomeruloid vascular proliferations evident; 1 point:only single glomeruloid vascular proliferations in thewhole tumor specimen detectable after meticulousscreening; 2 points: easily detectable multiple glomeru-loid vascular proliferations. Vascular clusters were

135Vascular Patterns in Glioblastoma: Evidence for Distinct Angiogenic Subtypes—Birner et al

Figures 1. A-D: Vascular patterns in glioblastoma. A-C (anti-CD34, immunoperoxidase):. Bizarre vascular patterns compriseglomeruloid vascular proliferations (A, original magnification �100), vascular garlands (B, original magnification �100) and vascu-lar clusters (C, original magnification �100). Classic vascular pattern (D, original magnification �200) is characterized by capillary-like microvascular sprouting resembling classic angiogenesis. Confocal laser scanning microscopy (figure panel on the right hand)confirms the neoangiogenic nature of classic vascular pattern: anti-CD34 decorated endothelium of delicate tumor microvessels co-expresses neoangiogenic markers CD105 and Id-1. E, F: Expression of angiogenic proteins in glioblastoma. Diffuse nuclearexpression of HIF-1� (E, original magnification �200) and strong cytoplasmic expression of VEGF (F, original magnification �300)as observed most commonly in glioblastomas with predominant classic vascular pattern. G-J: Glioblastoma with losses at chromosomes1p and 19q. One long-term surviving patient (>600 days) with typical plain histology of glioblastoma (G, HE stain, original magnifi-cation �200) and strong expression of EGFR (H, original magnification �300) showed 2:1 ratios on FISH analysis of chromosomes1p (I, original magnification �3000) and 19q (J, original magnification �3000), definitely indicating a combined 1p/19q deletion sta-tus. Figures I and J show the hybridization results on a single isolated tumor cell nucleus, respectively. Green signals: paracentromericprobes; red signals: subtelomeric probes.

scored in the same way as glomeruloid vascular prolif-erations. Vascular garlands: 0 point: no garlands evi-dent; 1 point: a single garland; 2 points: ≥2 garlands. Scor-ing points of glomeruloid formations, clusters andgarlands in the individual tumor were added (maximumscore of 6). Tumors without microvascular sprouting orwith a score of bizarre vascular formations >2 (this cut-off level was based on the median sum of bizarre vascularformations) were attributed to the group with predomi-nant “bizarre” angiogenesis. If microvascular sproutingwas evident, and the sum of bizarre vascular formationswas ≤2, a tumor was attributed to the group with pre-dominant “classic” angiogenesis.

Microvessel density (MVD), assessed by immunos-taining for CD34, was determined according to Weidner(41). The immunostained sections were scanned at lowmagnification (�40) and the tumor area with the highestdensity of distinctly highlighted microvessels (“hotspot”) was selected. MVD was then determined in the“hot spot“ of one section by counting all vessels at a totalmagnification of �200 within an examination area of0.25mm2 using an eye grid. Each stained lumen wasregarded as a single countable microvessel. If nolumen, but only a single CD34 positive cell was visible,this cell was also interpreted as a single microvessel. Totest reproducibility of assessment of MVD, we selectedrandomly 5 cases with predominant classic and 5 caseswith predominant bizarre vascular pattern and assessedMVD on a second section. Correlation of first and sec-ond assessment of MVD was high (p<0.001, r = 0.976,Spearman’s coefficient of correlation) and there was nosignificant difference between the two investigations (p= 0.754, Sign-test).

Assessment of MIB1 immunolabeling was per-formed by counting stained and unstained tumor cellnuclei, excluding vascular endothelial cells andhematogenous cells. For each specimen, 500 tumorcells were counted in fields showing the highest densi-ty of immunopositive tumor cells. Labeled nuclei wereexpressed as percentage.

A specimen was considered as positive for p53expression, when more than 50% of tumor cells showeddistinct nuclear staining (5). At EGFR- immunostaining,glioblastomas with ≥5% of tumor cells showing mem-brane associated and/or cytoplasmic protein expressionwere considered as positive (36). Apoptotic index wasdetermined as follows: In the field with the highestnumber apoptotic signals, the numbers of non-labeled andlabeled nuclei were counted at magnification �400 in anarea of 0.1725 mm2 using an eye grid, and the percent-age of apoptotic cells was calculated.

Nuclear expression of HIF-1� and HIF-2� (repre-senting the biologically active form of these transcrip-tion factors) in tumor cells was assessed as follows: 1.perinecrotic; 2. focal; 3. around patent vessels; 4.around vessels with thrombotic occlusions; and 5. diffuseexpression: stained cells diffusely dispersed through-out the whole tumor specimen (>10% of tumor cells). Pat-terns 1-4 were commonly observed in combination,while in case of diffuse anti-HIF staining, all other HIFstaining patterns were not clearly discernible and thuswere not assessed.

Expression of VEGF by tumors was judged as“weak” or “strong,” based on the portion of stainedtumor cells and the staining intensity.

Statistics. Kruskal-Wallis-test, Mann-Whitney-test,Chi-square test and Kappa statistic were used as appro-priate.

Overall survival was defined from the day of initialsurgery until death of the patient. Death from anothercause than glioblastoma or survival until the end of theobservation period were considered as censored obser-vations. Survival probabilities were computed as outlinedby Kaplan and Meier (15). Log-rank test and Cox pro-portional- hazards model were used for uni- and multi-variate analyses of overall survival. PostoperativeKarnofsky performance status (≥80 versus <80),patients age (>60 versus ≤60 years), extent of resection,and the angiogenic subtype (mainly “classic” or“bizarre” angiogenesis) were entered into Cox regression.The cut-off values for age and Karnofsky status weredefined according to the median values.

For all tests, a 2-tailed p-value of ≤0.05 was consid-ered as significant.

Results

Clinical features. One hundred fourteen patientswere included in our study. Median age at initial surgerywas 60 years (range 35-78 years). In 67 cases (58.7%)the tumor was resected subtotally, while in 37 cases(32.5%) total removal of the tumor was documented. In10 cases (8.8%), no information on the extent of resec-tion was available. Eighty-nine patients (78.1%)received adjuvant chemo- and radiotherapy, 3 patients(2.6%) only radiotherapy, and 1 patient (0.9%) onlychemotherapy. In 11 patients (9.7%) no adjuvant thera-py was administered, and no information on adjuvanttherapy was available in 10 patients (8.7%). Medianfollow-up time was 1106 days (range 5-1509 days).During this observation period, 90 patients (78.9%)


died of their disease, while 24 patients (21.1%) were lostto follow up or died of causes unrelated to glioblas-toma. Median postoperative Karnofsky performancestatus was 80 (range 50-100). Since only patients withvery low Karnofsky performance status received noadjuvant therapy, no analysis of survival with regard totherapy was performed.

Neovascularization. We could evaluate vasculariza-tion patterns on anti-CD34 immunostained sections in allbut 4 cases. (Analysis was not possible in the lattercases due to strong anti-CD34 labeling of tumor cells, inaddition to vascular staining.) As basic vascular pat-terns we observed bizarre glomeruloid/garland-like/clustered vascular formations (bizarre vascular pat-tern) on the one hand and capillary-like microvascularsprouting resembling classic angiogenesis (classic vas-cular pattern) on the other hand. To confirm the neoan-giogenic nature of classic vascular pattern, we per-formed confocal laser scanning microscopy (LSM) in 4selected cases. LSM showed that the endothelium ofthe anti-CD34 decorated delicate tumor microvesselsexpressed neoangiogenic markers CD105 (endoglin)

(21) and Id-1 (24) (Figure 1D), while endothelial cellsof microvessels in adjacent normal brain tissue showedno immunostaining for CD105 or Id-1. Analyzing vas-cular patterns more closely, we observed that there wasa variable prominence of bizarre and classic vascular pat-terns. We further had the impression that glioblastomaswith a low content of bizarre vascular pattern hadprominent classic vascular pattern, and vice-versa.Therefore we hypothesized that 2 angiogenic subtypesof glioblastoma may exist, the one with predominantbizarre angiogenesis and the other with predominantclassic type of angiogenesis. To test this hypothesis, weassessed vascular patterns using a semiquantitativescoring system (for definition of the scoring system, seeMethods section). Further, we performed correlationanalysis between bizarre and classic vascular patterns, andwe correlated the vascular scoring data with clinicaloutcome, microvessel density (MVD), and expression ofangiogenic and cell proliferation-related proteins (seebelow).

Score 1 and score 2 glomeruloid vascular structureswere found in 60 (54.5%) and 10 (9.1%) cases, respec-tively. Score 1 and score 2 vascular garlands were


classic angiogenesis (n = 29) bizarre angiogenesis (n = 81)

HIF-1� expressiondiffuse* 6 (21.4%) 5 (6.2%)around necrotic areas 18 (78.3%) 59 (80.8%)around thrombosed vessels 3 (13%) 13 (17.1%)around patent vessels 3 (13%) 11 (14.5%)focal 8 (34.8%) 27 (35.5%)

HIF-2� expressiondiffuse 0 0around necrotic areas 2 (6.9%) 1 (1.2%)around thrombosed vessels 0 0around patent vessels 0 0focal 2 (6.9%) 1 (1.2%)cytoplasmic 9 (31%) 13 (16%)in macrophages 9 (31%) 28 (34.6%)

Strong VEGF-expression* 12 (41.4%) 16 (19.8%)

Median apoptotic index* 2.72% (range 0-7.37%) 1.08% (range 0-7.12%)

Median microvessel density* 118 (range 35-266) 78 (range 10-407)

Median MIB1 labeling index 27.8% (range 3.3-72.3%) 26.9% (range 5.6-92.2%)

Median age (years) 55.8 (range37.5-77.9) 60 (34.9-76.4)

12-months survival rate* 53% 44%

Median survival time* 401 days 320 days

* significant difference between tumor subtypes (p≤0.05)

Table 1. Differences between tumors with predominant classic and bizarre angiogenesis.

observed in 48 (43.6%) and 8 (7.3%) patients, respec-tively. Score 1 and score 2 vascular clusters were foundin 74 (67.3%) and 6 (5.5%) patients, respectively. Themedian sum of all types of bizarre vascular formationswas 2 (range 0-4). There was a significant negativeassociation between the score of bizarre vascular struc-tures and microvascular sprouting (p<0.001. Mann-

Whitney test). Eighty-one patients (73.6%) were attrib-uted to the subtype with predominant bizarre vascular pat-tern and 29 patients (26.4%) were attributed to the sub-type with predominant classic vascular pattern (scoringcriteria for these subtypes, see Methods section). To testvalidity of the results, we re-analyzed all anti-CD34stained sections blinded to the scoring results. We clas-sified the cases on the basis of plain histologicalimpression either as mainly bizarre or classic vascular pat-terns. This review resulted in classification of 83 casesas predominant bizarre vascular patterns and 27 cases(23.7%) as predominately classic vascular patterns.Thus, results of plain histological assessment as comparedto the scoring system were discrepant in only 4/110cases. Statistically, correlation between the results ofthe scoring system and plain histological assessmentwas very high (p<0.001, Kappa: 0.904). For all furthercalculations we used results obtained with the scoring sys-tem.

Cell proliferation, apoptosis, and expression ofangiogenic proteins. Median MIB1 labeling index was26.7% (range 3.3-92.2%). Twenty-nine (25.4%) ofcases were considered as positive with regard to p53expression. At EGFR-immunostaining, 65 cases (57%)were considered as positive. Median apoptotic indexwas 1.35% (range 0-7.37%.). Analyzing all 114 tumors,diffuse expression of HIF-1� was observed in 11 cases(9.6%) (Figure 1E), while no expression of HIF-1� wasfound in 18 cases (15.8%). In the remaining 85 cases,HIF-1� expression directly adjacent to necrotic areas wasobserved in 81 cases (71.1% of all cases), around patentvessels in 14 cases (12.3%), around thrombotic vesselsin 17 cases (14.9%) and focal expression of HIF-1�was seen in 36 cases (31.6%). In a previous studyZagzag et al demonstrated on serial sections that areaswith focal HIF-1� immunoreactivity proved to bedirectly adjacent to necrotic areas (45). So in contrast todiffuse expression of HIF-1� all the focal staining pat-terns seem hypoxia-induced.

Anti-HIF-2� immunostaining showed hypoxia-related staining patterns whereas a diffuse staining pat-tern was not observed (Table 1).

Weak expression of VEGF was seen in 85 tumors(74.6%), while expression was strong in 29 cases(25.4%) (Figure 1F).

Correlation of histological features. Range of MVDwas 10 to 407 microvessels/field, with a median of 84.Correlating with vascular patterns, we observed thatMVD was significantly higher in patients with classic vas-


Figure 2. A. Mean microvessels/ field (microvessel density) +standard error (SE) of cases with predominant bizarre andclassic vascular pattern. B. Mean apoptotic index + standard error(SE) of cases with predominant bizarre and classic vascular pat-tern. C. Cumulative overall survival of glioblastoma-patientswith tumors with a) predominant classic vascular pattern (n=29);b) predominant bizarre vascular pattern (n = 81).

cular pattern (median 118, range 35-266 microvessels/field) as compared to patients with predominant bizarrevascular pattern (median 78, range 10-407 microves-sels/ field) (p = 0.026, Mann-Whitney test) (Figure 2A).In the group with classic vascular pattern, diffuseexpression of HIF-1a was more common (p = 0.031,exact Chi-square test) (Table 1) as was strong VEGF-expression (p = 0.027, Chi-square test) (Table 1), andthe apoptotic rate of tumor cells was significantly high-er (p<0.001, Mann-Whitney test) (Figure 2B, Table 1).There was no correlation of vascular pattern or MVD withMIB-1 labeling index or HIF-2� expression.

Cases with diffuse HIF-1� expression had: i) a lowerscore of bizarre vascular structures (median grade 1versus 2, p = 0.04, Mann-Whitney test); ii) more oftenmicrovascular sprouting (63.3% versus 30.6% of cases,p = 0.042, Chi-square test); and iii) stronger VEGF-expression (p<0.001, Chi-square test). There was noassociation of diffuse HIF-1� expression with apoptot-ic index, MIB1 labeling index, or MVD (p>0.05,Mann-Whitney test). We found no association of p53 sta-tus or EGFR- expression with predominant classic orbizarre vascular pattern (p = 0.889, p = 0.19, respective-ly; Chi-square test). There was also no association of vas-cular patterns with age of patients (p = 0.441, Mann-Whitney test).

Survival analysis. In univariate analysis (log-ranktest), high postoperative Karnofsky performance status(≥80 versus <80) (p = 0.0006) and young patients’ age(>60 versus ≤60 years) (p = 0.0304) correlated with pro-longed survival. Further, survival was significantlylonger in patients with predominant classic vascularpattern compared to those with predominant bizarre one(p = 0.0048, log-rank test) (Figure 2C). No influence ofapoptotic index (≤1.35% versus >1.35%) on survivalwas found (p = 0.65, log-rank test).

Diffuse HIF-1� expression had no influence on sur-vival (p = 0.89). In multivariate analysis of overall sur-

vival (Cox regression), a predominant classic vascularpattern and postoperative Karnofsky performance statusremained as independent prognostic factors (p<0.01)(Table 2). These results are based on assessment of vas-cular patterns (bizarre and classic patterns) using asemiquantitative scoring system (see Methods section).Repeating statistical analysis using vascular pattern cat-egories as obtained by plain histological assessment(see Methods section), results of uni- and multivariatesurvival analysis remained practically identical (datanot shown).

Among patients with predominant bizarre vascular pat-tern, 13 (16%) survived longer than 600 days (median 790days, range: 622-1106 days; 11 deaths). In the group withpredominant classic vascular pattern, 7 patients(24.1%) survived longer than 600 days (median 1189days, range: 657-1509 days; 4 patients are still alive657-1509 days after surgery).

The classic vascular pattern in our series associatedwith long-term survival may be considered as reminis-cent of oligodendroglioma. Therefore, we re-examinedin detail tumors of long-term survivors (>600 days)with predominant classic vascular pattern (n = 7). Onplain histology, none of the cases showed oligoden-droglial pattern but features typical of glioblastoma.Three cases showed strong anti-p53 labeling, 3expressed EGFR and one showed combined p53 andEGFR expression. FISH-analysis showed no loss ofchromosomes 1p or 19q in 6/7 cases (one case had 19qgain). In one patient, a 62-year-old female, we found com-bined 1p and 19q loss. This case showed a high prolif-eration rate (MIB1 labeling index: 35%), prominentEGFR-expression, diffuse immunostaining for HIF-1�and weak expression of VEGF. The duration of symptomsbefore surgery was 30 days, and at last follow-up, thepatient was alive 1354 days after surgery. Histology andFISH results of this case are shown in Figure 1G-J.


Factor p-value relative risk 95% interval of confidence

predominant classic versus bizarre angiogenesis 0.006 0.436 0.24-0.792

age (>60 versus ≤60 years) 0.184 -

postoperative Karnofsky performance status

(≥80 versus <80) 0.005 0.421 0.231-0.766

extent of resection 0.845 -

Table 2. Multivariate analysis of cumulative overall survival of 114 patients with primary glioblastoma (Cox regression).

DiscussionIn glioblastoma, prognosis is dismal and the effect of

conventional therapies is limited. Thus there is highneed and interest in novel therapies based on molecularinsights. Due to the prominent neovascularization,glioblastoma has become a strong candidate for anti-angiogenic treatment (28), and several experimentalanti-angiogenic therapy approaches for glial braintumors are currently under development (23, 37). In arecent phase II clinical trial using thalidomide as anti-angiogenic agent an antitumorigenic effect has beenreported in a subset of patients with recurrent highgrade gliomas (12). Nevertheless, possible beneficialeffect of anti-angiogenic therapies has remained a con-tentious issue (42). Detailed knowledge of clinico-pathologic aspects of neoangiogenesis in humanglioblastoma is a prerequisite for successful translationof anti-angiogenic therapy approaches into clinical use.Vascular patterns may have an important impact on theefficacy of anti-angiogenic therapy (3).

Vascular patterns in human and experimentalglioblastoma. We performed analysis of neovascular-ization in a large consecutive series of primary glioblas-tomas and observed as basic vascular patterns bizarreglomeruloid/garland-like/clustered vascular formations(“bizarre” vascular pattern) on the one hand and capil-lary-like microvascular sprouting resembling classicangiogenesis (“classic” vascular pattern) on the otherhand. There was a significant negative correlation ofthe prominence of bizarre and classic vascular patterns.In case of bizarre vascular pattern, cell growth seems tooutpace neovascularization, so progression of thesetumors seems not to depend on adequate vascularization.It seems unlikely that glioblastomas with such a vascu-lar pattern will profit from anti-angiogenic therapy. In caseof classic vascular pattern, cell growth seems to paral-lel neovascularization, indicating the pathobiologicalrelevance of angiogenesis in these glioblastomas. So itseems probable that glioblastomas with such an evenlydistributed and delicate vascular pattern are more like-ly to respond to an anti-angiogenic therapy. Interesting-ly, in GFAP-v-src transgenic mice, which have beenproposed as models for study of angiogenesis in humangliomas, mainly a “classic” vascular pattern isdetectable whereas bizarre vascular proliferations sim-ilar to human glioblastoma are not observed (40). Thisseems also to be the case in other transgenic gliomamodels (11). Due to the similar vascular architecture inglioblastomas with classic vascular pattern and experi-mental mouse gliomas, translation of successful exper-

imental anti-angiogenesis seems more probable tohuman tumors with predominant classic angiogenesisthan to those with mainly bizarre vascular patterns.However, the possible influence of vascular patterns onthe effect of anti-angiogenic therapy needs to be clari-fied in future anti-angiogenic therapy trials.

Glioblastomas with classic vascular pattern showedsignificantly better survival, and a predominant classicvascular pattern remained even an independent highly sig-nificant prognostic factor in multivariate analysis, pre-dicting survival as sensitive as a high postoperativeKarnofsky index. A possible explanation might be alower proliferative activity in glioblastomas with clas-sic vascular pattern, thus representing a less malignanttumor subgroup. Therefore, we compared MIB1 label-ing in glioblastomas with classic and bizarre vascular pat-terns. Statistical analysis did not disclose any differ-ence of MIB1 labeling between these two groups, thusarguing against differences in cell proliferation.

However, apoptosis was significantly more prominentin glioblastomas with classic vascular pattern and therewas a trend towards longer survival in patients withhigh apoptotic rate. On basis of our data, we cannotprovide a definite conclusion which factors are deci-sive for the differences in survival and rate of apoptosis.One possible explanation is a better access ofchemotherapeutic drugs to tumor cells and more effec-tive adjuvant radiotherapy in glioblastomas with classiccapillary-like vascular pattern. Another explanation isincreased apoptosis, possibly induced by HIF-1� over-expression: HIF-1� is known to exert a proapoptoticeffect (2) and in our series, HIF-1� expression and theapoptotic rate are more prominent in glioblastomaswith classic vascular pattern.

In oligodendroglial neoplasms, combined deletionof chromosomes 1p and 19q is associated withchemoresponsiveness and long-term survival (13). Theassociation of predominant classic vascular pattern andlong-term survival in our series may be considered asreminiscent of oligodendroglioma. However, none ofour cases showed oligodendroglial features on plainhistology and only 1 of 7 patients with classic vascularpattern and survival >600 days showed combined1p/19q loss. Combined 1p/19q loss in glioblastomawithout oligodendroglial features has been reportedrecently also by others (34).

HIF-proteins in glioblastoma. HIF-1 and 2� arekey factors in induction of neoangiogenesis (10). HIF-1� is considered to support tumor growth throughinduction of angiogenesis via transactivation of the


VEGF gene, as demonstrated in a variety of studies (8,14, 29). A direct association between microvessel den-sity and HIF-1� expression has been demonstrated in sev-eral human tumors (4, 6, 7, 45). In addition, HIF-1� isalso able to stimulate tumor progression by VEGF-independent mechanisms (30). There are 2 potentialfactors stimulating HIF expression, namely hypoxiaand nonphysiologic oncogenic stimuli (35). Few dataexist on expression of HIF proteins in glioblastomas: HIF-1� was considered to be mainly induced by hypoxia,based on immunostaining of 12 cases of glioblastoma,where the majority of positive cells was found aroundnecrotic areas (45). In addition to this finding, weobserved in our series diffuse HIF-1� expressionthroughout the whole tumor in 10% of cases. Thisobservation indicates that HIF-1� seems not to beinduced by hypoxia alone in these cases, but rather byoncogenic stimuli, as already demonstrated in a varietyof non-hypoxic tumors (39, 46, 48). Although no directassociation between microvessel density, apoptoticindex and diffuse HIF-1� expression was found in ourseries, these parameters were significantly elevated intumors with predominant classic vascular pattern. Aplausible interpretation of these data is that oncogenicupregulation of HIF-1�, evident by diffuse immunohis-tochemical pattern, may result in increased VEGF-expression, microvessel density, and apoptotic index ina subset of tumors with predominant classic vascular pat-tern.

HIF-2� is remarkably similar to HIF-1� with respectto the structure and regulation of activity by hypoxia (27,43). Only few data on HIF-2� expression in humanglioblastoma exist (39), and Zagzag et al hypothesizedthat HIF-2� protein might be more commonlyexpressed in glioblastomas than HIF-1� (45). Our databased on a large collective demonstrate that HIF-2�protein is expressed only in few tumor cells, withhypoxia-related pattern. On basis of our data it seemsunlikely that HIF-2� plays a primordial role in thepathophysiology of glioblastoma. Therapeutic strate-gies targeting HIF-proteins are currently under devel-opment (20, 22, 26, 38, 47). Due to the proangiogenic andproapoptotic role of HIF-proteins, the therapeutic effectof anti-HIF-strategies is difficult to predict. Possibly,such therapies might impede glioblastoma growth, or onthe contrary, even accelerate tumor growth because ofablation of the proapoptotic effect. Thus, translation ofanti-HIF strategies into clinical application will requirea cautious and well controlled procedure.

Conclusion. Altogether, results of our study providefor the first time firm evidence for distinct angiogenic sub-types of glioblastoma. These subtypes may respondvariably to anti-angiogenic therapies. Thus, future anti-angiogenic therapy approaches should take vascularpatterns and expression of angiogenic proteins intoaccount and compare therapy effects between angio-genic subtypes.

AcknowledgmentsThe help of Helen Turley, Helga Flicker, Ulrike

Köck, and Michaela Strohschneider was highly appre-ciated. This study was supported by the ScientificFunds of the Mayor of Vienna (Project-No. 2026) and byCCRI, St. Anna Children’s Hospital Vienna.

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vascular patterns in glioblastoma influence clinical outcome and associate with variable expression...

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