tenascin-c expression relates to clinicopathological features - lisa

Tenascin-C expression relates to clinicopathologicalfeatures in pilocytic and diffuse astrocytomas

C. Maris*, S. Rorive*, F. Sandras*, N. D’Haene*, N. Sadeghi†, I. Bièche‡, M. Vidaud‡,C. Decaestecker§¶ and I. Salmon*

Departments of *Pathology and †Radiology, Erasme University Hospital, and §Laboratory of Toxicology, Institute ofPharmacy, Université Libre de Bruxelles, Brussels, Belgium, ‡Laboratoire de Génétique Moléculaire, Faculté des SciencesPharmaceutiques et Biologiques, Université Paris V, Paris, France, and ¶Fonds National de la Recherche Scientifique,Brussels, Belgium.

C. Maris, S. Rorive, F. Sandras, N. D’Haene, N. Sadeghi, I. Bièche, M. Vidaud, C. Decaestecker and I. Salmon(2008) Neuropathology and Applied Neurobiology 34, 316–329Tenascin-C expression relates to clinicopathological features in pilocytic and diffuse astrocytomas

Aims: Tenascin-C (TN-C) is an extracellular matrix brainglycoprotein for which conflicting in vitro and in vivoresults are reported in the literature dealing with itsinvolvement in astrocytoma aggressiveness, in particularastrocytoma invasion. In view of these conflicting resultsand the lack of data available on low-grade astrocytomas,the present study focuses on pilocytic World Health Orga-nization (WHO) grade I, and diffuse WHO grade II astro-cytomas, that is, two histological entities associated withvery different invasive abilities. Methods: Using real-timereverse transcription polymerase chain reaction andimmunohistochemistry, we analysed the TN-C expressionin normal brain tissue as well as in a series of 54 pilocyticand 53 grade II astrocytomas. Conclusions: Our data onnormal brain showed that while TN-C is largely expressed

in supratentorial white matter, it was largely absent ininfratentorial white matter. Paralleling these observa-tions, we showed that TN-C expression in low-grade astro-cytomas similarly varies according to tumour site. Coxregression analysis evidenced that TN-C provided an inde-pendent prognostic value which is enhanced in the case ofgrade II astrocytomas for which positive TN-C expressionis associated with a higher risk of recurrence. We alsoanalysed TN-C expression specifically in vascular areas oflow-grade astrocytomas without demonstrating any prog-nostic value for this additional feature. Results: Similarlyto normal brain, low-grade astrocytomas exhibit varia-tions in TN-C expression with site, and this expression isassociated with an independent prognostic value in termsof recurrence.

Keywords: extracellular matrix, low-grade astrocytoma, multivariate analysis, prognosis, tenascin-C

Introduction

Astrocytomas can be grouped into histological entitiesassociated with very different clinical behaviour patternsand pathological findings, and are classified into fourcategories (grade I–IV) according to the World HealthOrganization’s (WHO) grading system [1,2]. Grade IVastrocytomas (glioblastomas) constitute the most aggres-

sive type, and their biological characteristics are widelystudied. While grade II diffuse astrocytomas constitutethe first step in the malignancy development continuumwithin the so-called group of diffuse astrocytic tumours(that is, WHO grade II–IV), grade I pilocytic astrocytomasare generally well delineated and do not progress to highergrades. However, aggressive progression may be observedin a number of pilocytic astrocytomas with an infiltrationpattern often leading to tumour recurrence, even thoughthese tumours do not necessarily share the biologicalfeatures of diffuse high-grade astrocytomas [2–4].

Correspondence: Isabelle Salmon, Departments of Pathology, ErasmeUniversity Hospital, Université Libre de Bruxelles, Brussels, Belgium.Tel: +322 553115; Fax: +322 554790; E-mail: [email protected]

316 © 2007 Blackwell Publishing Ltd

Neuropathology and Applied Neurobiology (2008), 34, 316–329 doi: 10.1111/j.1365-2990.2007.00898.x

mailto:[email protected]

Aggressiveness and poor prognosis in the case of patientssuffering from astrocytomas are mainly associated withthe ability of astrocytoma cells to invade the surround-ing parenchyma; this ability is modulated by the tumourenvironment, especially the extracellular matrix (ECM)[5]. In addition, the ECM plays a number of central rolesin other biological processes, such as proliferation andangiogenesis, which are involved in astrocytoma devel-opment [5–8]. While the composition and the roles ofthe ECM in the central nervous system (CNS) are not welldefined, various authors agree that the ECM of the brainis different from that of the other organs. While collagen,fibronectin and laminin are the predominant ECM com-ponents in non-CNS tissue, hyaluronan, proteoglycans,tenascin-C (TN-C) and thrombospondin are the principalelements in the CNS ECM [8,9]. TN-C is a glycoproteincharacterized by a hexameric structure. Its amino-terminus includes a series of cysteines and heptadrepeats, both of which are involved in multimerization.This terminal part is followed by 14.5 epidermal growth-factor-like and 17 fibronectin type III-like repeats. Finally,the carboxy-terminus consists of a COOH-terminal knobmade up of a sequence homology with the globulardomain of the b and g chains of human fibrinogen[10,11]. While TN-C is encoded by a single gene underthe control of a single promoter, a number of structur-ally and functionally different human TN-C isoforms aregenerated by the alternative splicing of the TN-C tran-script at the fibronectin type III repeat level [10,11]. Byregulating the adhesive and signalling properties of cells,TN-C is able to play a number of morphoregulatory rolesduring the processes of development and tissue remodel-ling as well as in disease [10–13]. However, conflicting invitro and in vivo results are reported in the literature con-cerning the role that TN-C plays in the different processessuch as cell adhesion, motility and invasion that lead toastrocytoma cell migration – see the Discussion [14–21].It should be emphasized that most TN-C-related studiesconcern high-grade astrocytomas. In fact, very limiteddata are available on low-grade astrocytic tumours, andin particular on pilocytic (WHO grade I) astrocytomas,which display very different behavioural patterns as com-pared with the diffuse (WHO grade II–IV) group. Thismotivated us to analyse the TN-C expression in normalbrain tissue as well as in a series of 107 low-grade astro-cytomas [by means of real-time reverse transcriptionpolymerase chain reaction (RT-PCR) and immunohis-tochemistry] and to focus on the potential involvement of

TN-C in the magnetic resonance imaging (MRI) aspectsof low-grade astrocytomas. To this end, we used multi-variate data analyses that enabled us: (i) to study thecase distribution in a multivariate (clinical and anato-mopathological) feature space in which the feature inter-actions were analysed (by means of Log-linear models);and (ii) to carry out multivariate prognosis analyses (bymeans of Cox regression).

Materials and methods

Clinical and histopathological data

The investigations using normal tissue were carried outon samples from five normal human post mortem brains(without neuropathological alterations) obtained within24 h of death. Six samples were taken from six differentareas: grey and white matter from the cerebral hemi-spheres (frontal lobes) and grey and white matter from thecerebellar hemispheres, the brainstem and the cervicalspinal cord. Three samples from each site were storedat -80°C for RT-PCR analyses and three others wereembedded in paraffin.

A series of 107 low-grade astrocytomas was investi-gated in parallel. This series consisted of archivalformalin-fixed and paraffin-embedded samples obtainedfrom the Laboratory of Pathology of the Erasme Univer-sity Hospital (Brussels, Belgium) and collected between1984 and 2005. As detailed in Table 1, all the cases wereclassified by two pathologists according to the WHO clas-sification [1,2]. Frozen tumour samples from astrocyto-mas were also available for RT-PCR analyses (see below).These samples were used subject to the approval of theUniversité Libre de Bruxelles Hôpital Erasme EthicsCommittee.

The available clinical data included patients’ ages andgenders, their tumour sites, the extent of their surgicalresections and their pre- and post-surgical adjuvant treat-ments and follow-ups, as detailed in Table 1.

The preoperative neuroimaging data (including fluidattenuated inversion recovery and enhanced T1-weightedimages when available) were retrospectively reviewed andenabled 100 astrocytomas to be classified as either well- orill-circumscribed (see MRI status in Table 1). Because ofthe absence of normal tissue in the available materials,histopathological infiltration could not be assessed for alarge number of cases and was thus not considered in thepresent study.

Tenascin-C expression in astrocytomas 317

© 2007 Blackwell Publishing Ltd, Neuropathology and Applied Neurobiology, 34, 316–329

The recurrences concerned patients having benefited ofa total or subtotal surgery, and were defined as casespresenting magnetic resonance imaging (MRI) evidenceof progression that required new surgery or adjuvanttreatments.

Immunohistochemistry

Five-micrometer-thick sections were submitted to stan-dard immunohistochemistry as previously detailed[22,23], with the immunohistochemical expression beingvisualized by means of streptavidin—biotin–peroxidasecomplex kit reagents (BioGenex, San Ramon, CA, USA)with diaminobenzidine/H2O2 as the chromogenic sub-strate. Counterstaining with haematoxylin concluded theprocessing. The TN-C expression was evidenced by meansof a murine monoclonal anti-TN-C antibody clone DB7

(Chemicon Int, Temecula, CA, USA; dilution 1:200).Figure 1 illustrates the antibody specificity (1:750 dilu-tion) proven by means of Western blot analysis usingpurified human TN-C protein (1.25 mg; Chemicon Int,Temecula, CA, USA; Figure 1 lane 3), 200 mg of proteinsfrom the normal supratentorial grey (lane 4) and white(lane 5) matter, and 50 mg of proteins from two glioblas-tomas (lanes 6 and 7). For immunohistochemistry pur-poses, a negative control was carried out by replacing theprimary TN-C antibody with non-immune serum (Dako,Glostrup, Denmark).

Evaluation of the immunohistochemicalTN-C expression

The TN-C immunostaining intensity was assessed by twoindependent observers (CM, SR) using standard light-microscopy. Where discrepancies were encountered, thecases were settled by consensus with a third observer (IS).In view of the staining patterns observed (see Results),TN-C immunostaining in astrocytomas was systematicallyevaluated in the ECM, distinguishing between a negative tolow expression level (labelled as ‘negative’) and a moderateto high one (labelled as ‘positive’). Additional investiga-tions were carried out in vascular areas. This enabledus to identify in a number of cases presenting a particularpattern of TN-C expression (illustrated in Results). Thisparticular pattern is defined by a major enhancement ofTN-C expression in the vessel walls and/or perivascularECM as compared with neighbouring tumour ECM, andwas mentioned as vascular enhancement (VE) in the fol-lowing. As detailed in the results, this latter evaluation waspossible with certainty in 50 pilocytic and 44 grade IIastrocytomas only. The two TN-C-related features werelabelled ECM TN-C and VE TN-C respectively.

Table 1. Clinicopathological data for 107 patients with low-gradeastrocytomas

Pilocytic Grade II

Number of patients 54 53Age: child/adult* 29/25 14/39Gender: male/female 29/25 30/23Sites

Cerebral hemisphere 12 22Diencephalon 7 2Cerebellum 20 9Brain stem 10 4Spinal cord 3 15Others† 2 1

MRI statusIll-circumscribed 23 46Well-circumscribed 24 7Not specified 7 0

Surgical resectionTotal 30 8Partial 22 40Biopsy 2 5

Adjuvant therapyPre-surgery 7 4Post-surgery 7 17

Follow-up (months)Range 1–335 1–348Median 44 39

Recurrence‡ (median delay) 14 (34) 20 (17)Death 4 8

The data present numbers of cases in the different categories exceptwhere other measurements are indicated (such as range).*Cut-off value of 18 years.†optic nerve, corpus callosum.‡cases with total or partial surgery.MRI, magnetic resonance imaging.

181.8 kDa

115.5 kDa

82.2 kDa

1 2 3 4 5 6 7

210 kDa

Figure 1. Western blot analysis showing negative control (lane 2);TN-C protein (lane 3); normal supratentorial grey matter (lane 4);normal supratentorial white matter (lane 5); and glioblastomas(lanes 6 and 7). TN-C, tenascin-C.

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Real-time RT-PCR

Real-time RT-PCR analyses were carried out on a seriesof tissue samples in order to evaluate the TN-C mRNAexpression. RNA extraction, cDNA synthesis and PCRreaction conditions have been described previously [23].After samples that did not satisfy the quality controls hadbeen excluded, the remaining series consisted of 40normal brain tissue samples (10 cerebral grey and 12white matter, 10 cerebellar grey and eight white mattersamples), nine pilocytic astrocytomas (four supratento-rial and five infratentorial including three cerebellar)and nine grade II astrocytomas (five supratentorial andfour infratentorial including one cerebellar). Quantitativevalues were obtained from the threshold cycle number(Ct value), at which the increase in the fluorescent signalassociated with an exponential growth of PCR productsbegan to be detected by the laser detector of the ABIPrism 7700 Sequence Detection System (Perkin-ElmerApplied Biosystems, Foster City, CA, USA) using the PEBiosystems analysis software according to the manufac-turer’s manuals. The precise amount of total RNA addedto each reaction mix (based on optical density) and itsquality were both difficult to assess. Because of this, wealso quantified the transcripts of RPLP0, an endogenousRNA control gene [24]. As previously detailed [23], theresults were termed ‘Ntarget’ and expressed as N-fold dif-ferences in target gene expression relative to the RPLP0gene. Primers for the RPLP0 and TN-C were chosenwith the assistance of the Oligo 5.0 computer program(National Biosciences, Plymouth, MN, USA). We carriedout searches in the dbEST and nr databases to confirmthe absence of single nucleotide polymorphisms and thetotal gene specificity of the nucleotide sequences chosenas primers. To avoid the amplification of contaminatinggenomic DNA, one of the two primers was placed at thejunction between two exons. The nucleotide sequences ofthe primers used were as follows: RPLP0-U (5′-GGC GACCTG GAA GTC CAA CT-3′) and RPLP0-L (5′-CCA TCAGCA CCA CAG CCT TC-3′) for RPLP0 trancripts (PCRproduct of 149 bp), TNCtot-U (5′-GAG GGT GAC CACCAC ACG CTT-3′) and TNCtot-L (5′-CAA GGC AGT GGTGTC TGT GAC ATC-3′) for total TNC transcripts (PCRproduct of 73 bp).

Gel electrophoresis was used to verify the specificity ofthe PCR amplicons. For each primer pair, we performedno-template control and no-reverse-transcriptase controlassays, which produced negligible signals (usually >40 in

Ct value), suggesting that the effects of the primer–dimerformation and genomic DNA contamination werenegligible.

Data analysis

All the statistical analyses were carried out using Statis-tica (Statsoft, Tulsa, OK, USA).

The first step was to study the relationships betweenpairs of qualitative variables by means of contingencytables. The significance of the potential associations wasevaluated by means of either the c2-tests or Fisher’s exacttests (in 2 ¥ 2 cases only).

The second step involved the log-linear analysis tech-nique, which is a multivariate extension of the c2-test ofindependence [25]. The major task was to establish thebest possible fit for the cell frequencies of a multiwaycontingency table by means of a log-linear model. Weidentified the simplest model that fitted the data (that is,explained the multivariate data distribution) by using amethodology similar to that detailed in [26] and basedon the ‘Automatic best model selection’ procedureincluded in the log-linear analysis module of the Statis-tica software. Finally, the standard Cox regression analy-sis was also used to fit an explanatory model to therelapse-free survival data. This method enabled thepossible simultaneous influence of several variables onthe survival period to be tested. In addition, the non-parametric Mann–Whitney test was used to compareindependent groups of quantitative values provided byreal-time RT-PCR.

Results

Expression of TN-C in the normal human brain

As shown in Figure 2, in the cerebral white matter area,there is a highly heterogeneous level of TN-C expressionwith a high level of immunopositivity, whereas the cortexdisplayed a low level of immunopositivity limited to thefirst layer and the external glia limitans (Figure 2A).While the leptomeningeal vessels (smooth muscle cells)showed moderate TN-C immunopositivity, the capillariesof the white and grey matter did not offer any immunopo-sitivity at all. In contrast, in the cerebellum, we observed acompletely different staining pattern consisting of theabsence of any expression in the white matter (Figure 2B),whereas the ECM of the molecular layer sometimes



Figure 2. Illustrations of immunohistochemical TN-C expression in normal human brains and astrocytomas. (A) Major difference instaining for TN-C of the grey and white matter in normal frontal lobes. (B) Absence of TN-C expression in the normal human cerebellum(molecular layer and white matter). (C) Diffuse and strong TN-C expression in the ECM of a supratentorial pilocytic astrocytoma in contrastto the weak or wholly absent TN-C expression in the ECM of an infratentorial pilocytic astrocytoma (D). Same variation of staining observedbetween a supratentorial (E) and an infratentorial grade II astrocytoma (F). Two patterns of vascular enhancement of TN-C expression(VE TN-C) in astrocytomas showing strong TN-C expression in vessels walls with (G) or without (H) perivascular enhancement. Originalmagnification ¥40 (A–B); ¥100 (G); ¥200 (C–F, H). ECM, extracellular matrix; VE TN-C, vascular enhancement tenascin-C.

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displayed a very weak level of expression. No TN-C immu-nopositivity was detected in the cerebellar vessels. Whilethe ECM of the brainstem and spinal cord specimens wasnegative, we did observe intracytoplasmic TN-C expressionin focal neurones. These results were globally confirmedat the mRNA expression level by means of quantitativeRT-PCR. As shown in Figure 3A, the TN-C mRNA levelsincreased in the cerebral white matter as compared withthe cerebellar (P = 0.047), with very low (if any) mRNAlevels in the grey matter regardless of its origin.

Expression of TN-C in astrocytomas

To ensure certainty in the detection of the presence orabsence of VE TN-C, the evaluation was carried out twiceby the same observer (IS), after excluding six casesbecause of the limited size of the available material. Of the101 remaining cases, 94 cases (50 pilocytic and 44 gradeII) were concordantly evaluated (that is, 93% of concor-dance), of which 33 cases exhibited VE TN-C as illustratedin Figure 2G,H. In the following, we restricted any analy-sis related to VE TN-C to these 94 concordant cases. Itshould also be noted that the distinction between the dif-ferent types of VE TN-C (observed in perivascular ECMonly, or vascular cells only, or both) did not add additionalinformation (data not shown). In addition, in the case ofvery strong VE TN-C, the identification of the vascular celltypes expressing TN-C was not possible.

A first analysis showed no significant dependencebetween the two TN-C-related features (ECM and VE

TN-C), revealing that these two features produceddifferent information on TN-C expression in low-grade astrocytomas. The following results detailedthe difference observed between these two features inrelation to the other clinicopathological featuresanalysed.

We also showed that there was no need to distinguishbetween the tumours of the patients submitted to pre-surgery adjuvant therapies (n = 11) and those of thepatients who had not undergone any such therapies (seeTable 1), because no difference in TN-C expression (ECMand VE TN-C) was evident across these two groups (datanot shown).

In relation to tumour type The data concerning the immu-nohistochemical TN-C expression in the low-grade astro-cytomas are detailed in Tables 2 and 3 and illustrated inFigure 2C–F, while the quantitative RT-PCR assessmentsare shown in Figure 3B. As detailed in Table 2, the totalpercentages of cases with positive TN-C expression intumour ECM slightly increased from 46% for the pilocyticto 68% for the grade II (P = 0.03). At the mRNA level, nodifference was observed between pilocytic and grade IIcases when their locations were not taken into account(data not shown). As explained below, this was not thecase when comparing infratentorial and supratentorialtumours.

Although VE TN-C seemed to be more prevalent in pilo-cytic (44%) than in grade II (25%) astrocytomas, thistendency was not significant (P = 0.08).

Figure 3. Real-time RT-PCR results of total TN-C mRNA expression in normal brain tissue (A) and low-grade astrocytoma samples (B). (A)The analyses were carried out on 40 samples of cerebral and cerebellar grey and white matter (10/12 cerebral grey/white matter, 10/8cerebellar grey/white matter). (B) The analyses were carried out on nine supratentorial (four pilocytic and five grade II) astrocytomas andnine infratentorial (five pilocytic and four grade II) ones. The data are expressed as means � SEM. RT-PCR, reverse transcription polymerasechain reaction; TN-C, tenascin-C; SEM, standard error mean.



In relation to site Because in the normal brain TN-C wasexpressed differently in terms of site, we analysed thepossible variations in astrocytoma TN-C expression inrelation to tumour site. The results shown in Table 2indicate that the slight increase in ECM TN-C expressionfrom the pilocytic to the grade II astrocytomas was simi-larly observed in the supratentorial and infratentorialastrocytomas. As also illustrated in Figure 2C–F, ECMTN-C was expressed in a significantly larger number of

supratentorial pilocytic (74%) and grade II (92%) astro-cytomas than their infratentorial counterparts (30% and50% respectively; P = 0.004 for pilocytic, P = 0.002 forgrade II and P < 10-6 over the total group of low-gradetumours). The ‘Others’ group containing only threecases was not taken into account in the statisticalanalyses.

The same tendency was observed at the mRNA level.When grouping the low-grade cases per location, a

Table 2. TN-C expression in low-grade astrocytomas

Pilocytic Grade II

ECM VE ECM VE

SitesSupratentorial

Hemisphere 83 (10/12) 64 (7/11) 91 (20/22) 6 (1/18)Diencephalon 57 (4/7) 50 (3/6) 100 (2/2) 0 (0/2)Subtotal 74 (10/19) 59 (10/17) 92 (22/24) 5 (1/20)

InfratentorialCerebellum 20 (4/20) 33 (6/18) 22 (2/9) 44 (4/9)Brain stem 40 (4/10) 50 (5/10) 50 (2/4) 75 (3/4)Spinal cord 67 (2/3) 33 (1/3) 67 (10/15) 33 (3/10)Subtotal 30 (10/33) 39 (12/31) 50 (14/28) 43 (10/23)

Others (1/2) (0/2) (0/1) (0/1)Total 46 (25/54) 44 (22/50) 68 (36/53) 25 (11/44)

The data are expressed as the percentages of cases exhibiting positive TN-C expression in ECM or VE. The exact ratios are also mentioned.TN-C, tenascin-C; extracellular matrix; VE, vascular enhancement.

Table 3. MRI status of low-grade astrocytomas in relation to their sites and TN-C expression

(A) Occurrence of ill-circumscribed astrocytomas in relation to tumour sites

Pilocytic Grade II

Supratentorial Cerebral hemisphere 44 (4/9) }44%95 (21/22) }96%Diencephalon 43 (3/7) 100 (2/2)

Infratentorial Cerebellum 19 (3/16) 56 (5/9)Brain stem 80 (8/10) }85%

75 (3/4) }89%Spinal cord 100 (3/3) 93 (14/15)Others (2/2) (1/1)

(B) Positive TN-C expression in relation to MRI status

Pilocytic Grade II

TN-C Expression ECM VE ECM VEWell-circumscribed 29 (7/24) 27 (6/22) 57 (4/7) 14 (1/7)Ill-circumscribed 61 (14/23) 62 (13/21) 70 (32/46) 27 (10/37)

The data in (A) are expressed as the percentages of ill-circumscribed tumours in each category (the exact ratios are also mentioned), while thedata in (B) are expressed as the percentages of cases exhibiting positive TN-C expression in ECM or VE (the exact ratios are also mentioned).TN-C, tenascin-C; MRI, magnetic resonance imaging; ECM, extracellular matrix; VE, vascular enhancement.

322 C. Maris et al.


decrease was observed in infratentorial as compared withsupratentorial cases (Figure 3B) without being signifi-cant. This decrease was probably due to the relative het-erogeneity detailed in Table 2 and the small number ofcases analysed.

Regarding VE TN-C, the pilocytic astrocytomas did notexhibit any significant variation in terms of tumour site(Table 2). This contrasts with the significant variations(P = 0.005) evidenced in grade II astrocytomas for whichVE was observed in infratentorial locations (including thecerebellum) almost exclusively (see Figure 2F).

In relation to patients’ ages We also investigated whetherpaediatric vs. adult astrocytomas were characterized bydifferent TN-C expression levels. We observed that grade IIastrocytomas in children generally had lower levels ofECM TN-C expression than those in adults (P = 0.006). Wedid not observe this relation in the case of the pilocyticastrocytomas (P = 0.10). No variation in VE TN-C wasevident between paediatric and adult astrocytomas.

In relation to the MRI status Finally, we focused onthe MRI status (well- vs. ill-circumscribed). The resultsdetailed in Table 3A show the variations in the MRI statusof the low-grade astrocytomas in relation to their sites,while bringing out the differences between the supraten-torial and the infratentorial sites where the cerebellar sitesmust be distinguished. The ill-circumscribed pilocytictumours were found particularly in the brainstem and thespinal cord (85%), were less frequently observed in thesupratentorial locations (44%) and even less so in the cer-ebellum (19%) (P = 0.002). As expected, the diffuse gradeII tumours were generally ill-circumscribed regardless ofsite (96% of supratentorial cases and 89% of brainstemand spinal cord ones), except in the cerebellum where only56% were ill-circumscribed (P = 0.009).

When the pilocytic and grade II astrocytomas weregrouped, a significantly higher number of ECM TN-C-positive cases were shown in the group of the ill-circumscribed tumours (66% vs. 35% for thewell-circumscribed astrocytomas, P = 0.005). As detailedin Table 3B, this relation was essentially due to the pilo-cytic astrocytomas (61% vs. 29% P = 0.04), for which asignificant association was also evident between VE TN-Cand the MRI status (62% vs. 27%, P = 0.03). In contrast,no significant relation was demonstrated between VETN-C and the MRI status in grade II astrocytomas (or aftergrouping the pilocytic and grade II astrocytomas).

Analysis of the multivariate interactionsbetween TN-C expression, tumour grades, sitesand MRI status and patients’ ages

The results reported above led us to consider that interde-pendencies exist between the different variables, particu-larly in the case of low-grade astrocytomas. We decided toperform a multivariate analysis in order to analyse theseinterdependencies systematically. We therefore carriedout a first log-linear analysis on the five-dimensional con-tingency table, cross-classifying ECM TN-C expression,patients’ ages (child vs. adult), tumour grades (I vs. II),sites (distinguishing between supratentorial, cerebellarand other infratentorial tumours) and MRI status (ill- vs.well-circumscribed). For the set of 97 tumours so con-cerned (located in the four major sites and for which theMRI status were available), the log-linear analysis wasable to test for statistical significance in the case of eachpossible interaction between two or more features; Table 4describes the results obtained. To begin with, we evaluatedthe order of the significant feature interactions (2, 3 or 4)that have to be taken into account in a log-linear model toexplain the data correctly (that is, without any significantloss of information). The results shown in Table 4Aindicate that only the two-feature interactions were sig-nificant. This confirms both the presence of a degreeof interdependence between pairs of features and theabsence of more complex relationships. Second, the infor-mation provided by the different feature interactions wasanalysed by evaluating the partial and marginal asso-ciations, as detailed in Table 4B. Briefly, the partial asso-ciation between two features evaluates the loss of infor-mation due to the exclusion of the feature interaction ofinterest from a complete log-linear model of order 2(including all the effects due to the individual features andthe two-feature interactions). In addition, the marginalassociation between two features evaluates the gain ofinformation by adding this particular interaction to a log-linear model including only the effects of order 1 (due tothe individual features). The results (detailed in Table 4B)showed significant (partial and/or marginal) associationsfor each pair of features except between tumour grade andsite (interaction 24). It should be noted that of all thepartial associations involving ECM TN-C expression (inter-actions 12–15), only one was significant, that is, interac-tion 14 between ECM TN-C expression and tumour site.This means that failing to take account of astrocytomasites in the evaluation of ECM TN-C expression leads to a



highly significant loss of information (P = 0.002). Thebest (that is, the simplest) log-linear model identifying themain significant effects able to explain the data efficiently(without any loss of information) was then generatedfrom an initial model (see Table 4B). This best model con-sisted of only the feature interactions between ECM TN-Cexpression and the patient’s age (interaction 13) ortumour site (interaction 14), MRI status and astrocytomagrade (interaction 25) or location (interaction 45), andastrocytoma grade and the patient’s age (interaction 23).

These five interactions were thus necessary and sufficientto explain the data distribution in the five-dimensionalcontingency tables.

Figure 4 summarizes the information provided by thislog-linear analysis, showing the direct and indirect linksso evidenced between the features. This clarified the datareported in the previous section by indicating the presenceof a direct and essential association between ECM TN-Cexpression and the astrocytoma sites. The influence ofpatient’s age also seems to be an essential factor that has

Table 4. Log-linear analysis of the multivariate interactions between features

(A) Fitting of all K-feature interactions

K-feature interaction Degrees of freedom Max.Lik. c2 P-value

1 6 19.74 0.0032 14 75.05 <10-6

3 16 11.76 0.764 9 5.51 0.795 2 0.30 0.86

(B) Partial and marginal association – identification of the best model

EffectDegrees offreedom

Partial Ass.c2

Partial Ass.P-value

Mrg. Ass.c2

Mrg. Ass.P-value

Initialmodel

Bestmodel

Order 11 1 1.86 0.17 1.86 0.172 1 0.41 0.52 0.41 0.523 1 4.40 0.04 4.40 0.04 X4 2 2.80 0.25 2.80 0.255 1 10.27 0.001 10.27 0.001 X

Order212 1 0.87 0.35 4.93 0.03 X13 1 3.64 0.06 10.23 0.001 X X14 2 13.00 0.002 20.48 0.00004 X X15 1 1.31 0.25 7.54 0.006 X23 1 2.44 0.12 6.63 0.01 X X24 2 0.03 0.98 3.33 0.1925 1 8.78 0.003 14.01 0.0002 X X34 2 3.09 0.21 10.01 0.007 X35 1 0.55 0.46 6.46 0.01 X45 2 9.30 0.01 16.23 0.0003 X X

(A) Tests on the K-factor interactions (K = 1, 2, 3 or 4). A P-value <0.05 indicates that the corresponding interaction is significant. (B) Features:1, ECM TN-C expression; 2, tumour grade; 3, patient’s age; 4, tumour site; 5, MRI status. The different interactions are denoted by thejuxtaposition of the feature numbers. The partial association between the two feature i and j (denoted by ij) is computed by comparing the fit(that is, evaluating the c2 difference) of the complete model that includes all the two-way interactions with that of the model that excludes theinteraction between features i and j. The marginal association between the two features i and j is computed by comparing the fit of the model thatincludes all the main effects (that is, order 1) with that of the model obtained after the addition of the interaction between feature i and j only.The (marginal or partial) P-values <0.1 identify the features and interactions included in the initial log-linear model to fit the data. The bestmodel is the simplest model that is able to explain the data efficiently (that is, without any significant loss of information).Max.Lik., maximum licelihood; Partial Ass., partial association; Mrg. Ass., marginal association; ECM, extracellular matrix; TN-C, tenascin-C;MRI, magnetic resonance imaging.

324 C. Maris et al.


to be taken into account. This contrasts with the absenceof any direct relation between MRI status and ECM TN-Cexpression.

In a second log-linear analysis, we simply replaced theECM TN-C feature by the VE TN-C one. As seen for theinitial analysis, the best log-linear model generated onthese data was of order 2 (data not shown). In contrast,this model did not retain any interaction involving VETN-C but included it as an individual feature (that is, effectof order 1). However, it should be noted that significantand almost significant partial associations were, respec-tively, observed between VE TN-C and tumour grade(P = 0.03), and between VE TN-C and MRI status(P = 0.05). The actual importance of these associationshas to be confirmed on a larger series. In fact, the two-feature interactions retained in the best model resultingfrom the second analysis involved the other features(tumour grade, patient’s age, tumour site and MRI status)and confirmed the results already reported in Figure 4.

Prognostic impact of TN-C expression inlow-grade astrocytomas

We were not able to demonstrate any prognostic value forVE TN-C. However, for ECM TN-C ,while no correlationwas observed for pilocytic astrocytomas (P = 0.33;Figure 5A), positive ECM-TN-C expression was associatedwith a higher risk of recurrence in grade II astrocytomas(P = 0.07; Figure 5B).

In order to test the prognostic contribution of thismarker in the presence of standard clinical variables,multivariate Cox regression analyses were performed. Theresults confirmed an independent prognostic value for

ECM TN-C expression in the series of low-grade astrocyto-mas, which was also enhanced in the case of grade IIastrocytomas (see Table 5). We also observed that tumoursite did not add any prognostic information to the modelsdescribed in Table 5 (data not shown).

Discussion

Tenascin-C antibodies are presented as promising agentsin the design of treatment protocols for different types ofsolid cancers, including gliomas [27–30]. As explained byBrack et al. [27], this approach is motivated by the factthat antigens preferentially expressed in modified tumourECM are ideal targets for tumour-targeting applications.However, this type of application requires a detailedknowledge of the distribution of the antigen expression innormal and tumoral tissue, taking into account the highlevel of heterogeneity encountered in cancers.

While TN-C expression in the normal brain was rarelydetected in the first studies [10,31–33], most authors nowdescribe TN-C expression as being restricted to the ECM. Itshould be noted that only limited information is availableregarding normal brain tissues, and for a few studies peri-tumoral tissue was used as a point of normal reference bydifferent authors [15, 32–34]. This approach was avoidedin the present study because of the impossibility ofexcluding the TN-C secreted by astrocytoma cells migrat-ing in peritumoral areas (or by normal cells, such asendothelial ones, reacting in the tumour environment).The discrepancy in the data on the TN-C expression in thenormal brain could also be explained by the great com-plexity of the CNS. In fact, very limited data are availableon the actual distribution of TN-C expression across thedifferent structures of the normal brain. As previouslyshown by a few authors [18,19], the TN-C expression inthe normal brain is heterogeneous and increases in thewhite matter as compared with the grey matter. Our studyreveals that this heterogeneity is greater than supposedand is related to specific tissue sites. Indeed, we observedvery few, if any, cases of TN-C expression in the cerebellarwhite matter, although a readily discernible amount ofTN-C is present in the cerebral white matter.

In the case of astrocytomas, it is clear that TN-C expres-sion is heavily increased in glioblastomas as comparedwith normal tissue [15,17,18,34,35]. All the authors alsoagree that TN-C expression in ECM increases from low- tohigh-grade astrocytomas [15,17,18,34]. However, in thematter of TN-C involvement in astrocytoma aggressive-

Figure 4. Illustration of the direct links evidenced between thedifferent features analysed in the best log-linear model fitted to thedata (see Table 4 and text). Of these, the thick lines indicate thelinks associated with significant partial associations, that is,particularly required to explain the data.



ness, conflicting data have been reported in the literature,leading different authors to come to opposing conclusionsconcerning the roles played by TN-C in astrocytoma cellmigration, that is, a stimulating effect [14,15] vs. aninhibiting one [16]. Similarly, in vitro investigations,which performed migration assays, reported either astimulating effect [5,11] or an inhibiting one[8,15,21,36].

In view of these conflicting results, we focused ouranalysis on pilocytic and grade II astrocytomas, because

these two histological entities are associated with verydifferent invasive abilities [37]. As recently shown, thesetwo entities have different molecular profiles with respectto adhesion-, ECM- and invasion-related genes [38]. Inthe present series, neuroimaging evaluation revealed thatwhile a large majority of the grade II cases seemed to beinvasive, about half of the pilocytic cases displayed ill-circumscribed aspects.

Our monovariate analyses revealed that the ECM TN-Cexpression in the pilocytic and/or grade II astrocytomas

1.0

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Figure 5. Kaplan–Meier curves evidencing the relationships between the ECM expression of TN-C (positive/negative) and therecurrence-free survival periods for pilocytic astrocytomas (A) and grade II astrocytomas (B). The dots symbolize the recurrences and thecrosses represent the relapse-free cases respectively. ECM, extracellular matrix; TN-C, tenascin-C.

326 C. Maris et al.


varied according to the tumours’ sites, the patients’ agesand the MRI status. Multivariate log-linear analyses werethus required to evaluate the actual interdependencebetween the different features and to identify whichfactors actually are related to the ECM TN-C expression inthe astrocytomas. This approach has two main advan-tages. The first is that it provides a systematic approach tothe analysis of complex multidimensional tables, whilethe second is that it enables the relative importance of thedifferent effects (such as feature interactions) to be judged.This helped us to clarify that ECM TN-C expression inlow-grade astrocytomas is indirectly related to the MRIinfiltration status and directly related to the tumours’location. It is important to note that this model character-izes low-grade astrocytomas, which are well known fortheir occupation of both cerebellar and cerebral sites incontrast to high-grade astrocytomas, which are more fre-quently located in the cerebral hemispheres.

According to our hypothesis, the lack of TN-C in thenormal cerebellar ECM in contrast to its strong expressionin the normal cerebral ECM leads to very different tumoralECMs. Indeed, we have shown that a greater number ofboth supratentorial pilocytic and grade II astrocytomasexhibit ECM TN-C expression as compared with infraten-torial ones. It is known that high-grade astrocytoma cellsare able to secrete TN-C and thus strongly modify the ECMcomposition [6]. Our data suggest that low-grade astrocy-tomas do not have the same ability, and this may wellreflect on cell migration.

While future research should confirm these results on alarger series, our data suggest that VE TN-C could be asso-ciated with the MRI status, especially in the case of pilo-cytic astrocytomas. We also agree completely with Zagzaget al. [15], who emphasized that pilocytic astrocytomasdisplay an increase in perivascular TN-C enhancement,and especially around hyperplastic vessels. As reported inthe literature, endothelial cells are able to secrete TN-C[39], and the detection of TN-C in the vascular areas ofastrocytomas suggests a functional role played by TN-C inangiogenesis [15,17,18] and, more particularly, in themigration of endothelial cells [36]. The literature showsthat both glioma angiogenesis and invasion are invasiveprocesses sharing common mechanisms of regulationthat can be simultaneously inhibited by naturally occur-ring factors [15,17,18]. This may (at least partly) explainthe association between VE TN-C and the MRI statussuggested by our data. However, we did not succeed inshowing any prognostic value associated with VE TN-C forwhich the conflicting results reported in the literatureconcerned small series that did not include pilocytic astro-cytomas [15,17,18]. In contrast, ECM TN-C expressionwas associated with an independent prognostic value, par-ticularly in the case of grade II astrocytomas. This agreeswith another result associating ECM TN-C immunoposi-tivity with shorter survival for patients with glioblastomas(grade IV) [34].

In conclusion, similarly to normal brain, low-gradeastrocytomas exhibit variations in TN-C expression with

Table 5. Cox regression analysis

Model/P-value Variable b P-value

All pilocytic and grade II cases P = 0.00004 ECM TN-C 0.94 0.02Grade 0.73 0.08Age -0.04 0.005Surgery -1.24 0.03Adjuvant treatment 0.74 0.05

Grade II cases P = 0.002 ECM TN-C 1.93 0.006Age -0.06 0.002Surgery -0.70 0.37Adjuvant treatment 0.58 0.26

The ‘Model/P-value’ indicates the overall level of significance of the model. Except ‘Age’, which is a quantitative variable, all the others arebinary. ECM TN-C distinguishes between negative and positive expression, grade between pilocytic and grade II astrocytomas, surgery betweentotal and subtotal, and adjuvant treatment between absence and presence. The equation at the basis of the Cox Regression model is anexponential function of a linear combination of the variables considered, where b indicates the coefficient of each variable in the linearcombination. The associated P-value is a measure of the level of significance of the contribution of each variable to the model (and leads to theconclusion that b is significantly different from zero). If P < 0.05, the feature is associated with a significant prognostic value independently ofthe other parameters taken into account.ECM, extracellular matrix; TN-C, tenascin-C.



sites, and this expression is associated with an indepen-dent prognostic value in terms of recurrence. However,additional information could be provided by the currentlylacking analysis of different TN-C isoforms, especially inthe case of normal brain and low-grade astrocytomas. Tothe best of our knowledge, only one study mentions theabsence of large TN-C isoforms in these two categories oftissue as opposed to their presence in high-grade astrocy-tomas [34]. In fact, the scant availability of commerciallyavailable antibodies against alternatively spliced domainsnow limits immunohistochemical evaluation.

Acknowledgements

We are grateful to Ms Nathalie Watteau for secretarialsupport and Ms Blair Jenkins for help in preparing themanuscript. This work was carried out with the support ofgrants awarded by the Fonds Yvonne Boël (Brussels,Belgium).

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Received 25 July 2007Accepted 27 July 2007



tenascin-c expression relates to clinicopathological features - lisa

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