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Research article

Increased expression of EphA7 correlates with adverse outcome = in=20 primary and recurrent glioblastoma multiforme patients

Lin-Fang = Wang¹^,3=20 ,=20 Emmanouil Fokas¹ ,=20 Janko Juricko¹ ,=20 An You¹ ,=20 Frank Rose¹ ,=20 Axel Pagenstecher² ,=20 Rita Engenhart-Cabillic¹ and = Han-Xiang An¹

¹Department of Radiotherapy and Radiation Oncology,=20 Philipps-University Marburg, Baldingerstr. D-35043 Marburg, = Germany

²Department of Neuropathology, Philipps-University = Marburg,=20 Baldingerstr. D-35043 Marburg, Germany

³Department of Emergency Surgery, Union Hospital, = Tongji=20 Medical College, Huazhong University of Science and Technology, = 430030=20 Wuhan, People's Republic of China

=20 author email=20 corresponding author email

BMC Cancer 2008, = 8:79doi:10.1186/1471-2407-8-79

The electronic version of this article is the complete one and = can be=20 found online at: http://www.biomedcen= tral.com/1471-2407/8/79

Received:	8 August 2007
Accepted:	25 March 2008
Published:	25 March 2008

=C2=A9 2008 Wang et al; licensee BioMed Central Ltd.
This is = an Open=20 Access article distributed under the terms of the Creative Commons = Attribution License (http://creativecommon= s.org/licenses/by/2.0),=20 which permits unrestricted use, distribution, and reproduction in = any=20 medium, provided the original work is properly cited.

Abstract

Background

Malignant gliomas are lethal cancers, highly dependent on = angiogenesis=20 and treatment options and prognosis still remain poor for patients = with=20 recurrent glioblastoma multiforme (GBM). Ephs and ephrins have = many=20 well-defined functions during embryonic development of central = nervous=20 system such as axon mapping, neural crest cell migration, = hindbrain=20 segmentation and synapse formation as well as physiological and = abnormal=20 angiogenesis. Accumulating evidence indicates that Eph and ephrins = are=20 frequently overexpressed in different tumor types including GBM. = However,=20 their role in tumorigenesis remains controversial, as both tumor = growth=20 promoter and suppressor potential have been ascribed to Eph and = ephrins=20 while the function of EphA7 in GBM pathogenesis remains largely=20 unknown.

Methods

In this study, we investigated the immunohistochemical = expression of=20 EphA7 in a series of 32 primary and recurrent GBM and correlated = it with=20 clinical pathological parameters and patient outcome. In addition, = intratumor microvascular density (MVD) was quantified by = immunostaining=20 for endothelial cell marker von Willebrand factor (vWF).

Results

Overexpression of EphA7 protein was predictive of the adverse = outcome=20 in GBM patients, independent of MVD expression (p =3D 0.02). = Moreover, high=20 density of MVD as well as higher EphA7 expression predicted the = disease=20 outcome more accurately than EphA7 variable alone (p =3D 0.01). = There was no=20 correlation between MVD and overall survival or recurrence-free = survival=20 (p > 0.05). However, a statistically significant correlation = between=20 lower MVD and tumor recurrence was observed (p =3D 0.003).

Conclusion

The immunohistochemical assessment of tissue EphA7 provides = important=20 prognostic information in GBM and would justify its use as = surrogate=20 marker to screen patients for tyrosine kinase inhibitor = therapy.

Background

The incidence of brain tumors worldwide is about 7 in 100,000 = per year=20 [1,2]. = Glioblastoma=20 multiforme (GBM), the most aggressive tumor among malignant = gliomas, is=20 the most common primary brain tumor in adults and represents a = significant=20 source of cancer-related death. GBM usually recurs despite the = most=20 aggressive treatment via surgical resection of the tumor followed = by=20 radiation and/or chemotherapy [1,2]. = The poor=20 prognosis of patients with GBM (median survival ranging from 9 to = 12=20 months, 5-year survival rate close to 0%) mandates the exploration = of=20 novel molecular mechanisms that might contribute to the = pathogenesis of=20 this disease and its resistance to therapy with the purpose of = therapeutic=20 targeting [1-3].

Receptor tyrosine kinases (RTKs) are known to be important = regulators=20 of cellular growth controlling cell proliferation, differentiation = and=20 migration [4,5]. = The Eph=20 receptors and their ligands, ephrins, represent the largest known = family=20 of RTKs. Their role has been largely studied during the = development of=20 nervous system. They are involved in the development of central = nervous=20 system, including axon guidance, axon fasciculation, neural crest = cell=20 migration, hindbrain segmentation, vasculogenesis and neuronal = cell=20 survival during embryonic development [6-13]. = Eph=20 receptors and ephrin ligands are classified into A and B = subfamily, on the=20 basis of their sequence, homologies, structures, and binding = affinities.=20 EphA receptors bind the glycosylphosphatidylinositol = (GPI)-anchored=20 ephrin-A ligands, whereas EphB receptors bind the transmembrane = ephrin-B=20 ligands, whose cytoplasmic domain is capable to engage in various=20 signaling activities; an exception is the EphA4 receptor that = binds=20 ephrin-B2 and ephrin-B3 as well as ephrin-A ligands [14-16].=20 Moreover, these RTKs have the ability to induce both forward and = reverse=20 (bi-directional) signaling between adjacent interacting cells.

To date, various studies have investigated the involvement of = the=20 Eph-RTKs in several pathogenetic processes in the nervous system. = EphB2=20 and ephrin-B2 signaling participate in the glial scarring process = after=20 spinal cord trauma [17]. = The=20 phosphorylation ratio of R-Ras was closely linked to the = phosphorylation=20 ratio of EphB2 in glioblastoma tissues [18].=20 Additionally, the phosphorylation ratio of EphB2 is an important = mechanism=20 that mediates glioma cell migration and invasion [19]. = Ephrin-B2=20 and EphB4 were overexpressed by endothelial cells of human = malignant=20 gliomas [20]. = Ephrin-B3=20 was also demonstrated as an important factor regulating glioma = cell=20 invasion through Rac1 GTPase [21]. = EphA2=20 protein was overexpressed in GBM and anaplastic astrocytoma = tissues and=20 was identified as a novel target for the development of glioma = vaccines [22,23]. = Another=20 group confirmed overexpression of EphA2 expression in GBM cells, = probably=20 through decreased interaction between EphA2 receptor and its = inhibitory=20 ligand ephrin-A1 in malignant cells [19].

EphA7 (formerly known as Mdk1/Ebk/Ehk) is highly conserved in=20 vertebrates from fish to human [24]. = It is=20 widely expressed in embryonic tissues, especially developing = central=20 nervous system [25]. = EphA7=20 cooperates with other EphA receptors in cell signaling, but in = contrast to=20 other Eph receptors, it contains two developmentally regulated = isoforms: a=20 full-length version containing the intracellular tyrosine kinase = domain=20 and a truncated form that lacks this domain [26].=20 Immunoreactivity for the full-length wild type receptor is found = in all=20 cell populations expressing EphA7 mRNA in mouse embryo heads and=20 developing brain, while the truncated EphA7 is absent in the = embryos.=20 Interestingly, both isoforms show striking distributions in adult = mouse=20 brain [27]. = The=20 full-length EphA7 is strongly expressed in neuropil; in contrast = the=20 truncated EphA7 is conspicuous on cell bodies and proximal = dendrites of a=20 limited number of neuronal types [28]. = The=20 truncated form of EphA7 acts as a dominant negative antagonist,=20 suppressing tyrosine phosphorylation of the full-length EphA7 = receptor and=20 shifts the cellular response from repulsion to adhesion. = Additionally,=20 EphA7 is probably essential during closure of the neural folds, = since=20 EphA7-null mice displayed lack of the neural folds resembling = anencephaly=20 in man [29]. = Moreover,=20 EphA7 has been identified as an important molecular cue expressed = after=20 spinal cord injury, implicated in glial apoptosis [30]. = Recent=20 work indicated EphA7 as an important mediator of neural progenitor = apoptosis during brain development [31]. = However,=20 little is currently known about its role in brain tumor = angiogenesis and=20 pathogenesis.

In the present study, we investigated the immunohistochemical=20 expression of EphA7 and correlated it with clinical pathological=20 parameters and tumor vascularity. We provide evidence that EphA7 = is=20 overexpressed in GBM and suggest that this receptor might be used = as a new=20 diagnostic and prognostic marker for further Eph/ephrin targeted = molecular=20 cancer therapy.

Methods

Tissues

Tumor samples of 32 patients with histologically confirmed GBM, = WHO IV,=20 (26 primary GBM, 6 recurrent GBMs and 10 normal brain samples) = were=20 obtained from the Department of Neuropathology, Marburg University = Hospital, Germany. Approval for immunohistochemical study conduct = in this=20 GBM tissue bank had been obtained by the university authorities = together=20 with the signed consent of the patients. The patients underwent = surgery=20 and received adjuvant radiation therapy combined with chemotherapy = using=20 the schema ACNU and VM-26. The first follow-up occurred 6 weeks = after=20 therapy was completed. Subsequent follow-ups were scheduled every = 3=20 months. In addition to clinical investigations and monitoring of = indices=20 of recurrence, a radiological examination was performed to detect = possible=20 relapses. Disease progression was defined according to WHO = criteria by=20 either an increase of at least 25% in tumor size or any new tumor=20 identified by CT or MRI scan. Normal brain samples, which included = cortex=20 and white matter, were obtained from autopsy cases without any = evidences=20 of brain tumor or other brain disease. Totally, five cases of male = and=20 five cases of female (age ranging from 27 to 70 years, average: = 48.6=20 years) were obtained.

Immunohistochemistry

Immunohistochemical studies were performed on formalin fixed,=20 paraffin-embedded tissue. Samples slides were passed through a = sequence of=20 Roti-histol (Carl Roth, Karlsruhe, Germany) and graded alcohol and = then=20 rinsed in phosphate-buffered saline (PBS). After rinsing with PBS, = the=20 slides were treated with 3% hydrogen peroxide in PBS for 15 min at = room=20 temperature in order to abolish endogenous peroxidase activity.=20 Subsequently, the slides were treated with 5% blocking serum for 1 = hour.=20 Following this, slides were incubated overnight at 4=C2=B0C with a = rabbit=20 anti-human EphA7 polyclonal antibody (H-55) against amino acids of = human=20 EphA7 (1:100 dilution; Santa Cruz Biotechonology, Heidelberg, = Germany), or=20 rabbit anti-human Factor VIII (von Willebrand Factor, vWF) = polyclonal=20 antibody (1:400 dilution; Dako Cytomation, Carpinteria, CA). In = negative=20 controls, the primary antibody was replaced with 1 =C3=97 PBS. The = signal was=20 enhanced by using biotinylated polyclonal goat-anti-rabbit IgG = with=20 streptavidin-HRP (Dako Cytomation, Carpinteria, CA) for 30 = minutes. The=20 colour was developed after 5 minutes incubation with = 3,3-diaminobenzidine=20 (DAB) solution and sections were weakly counterstained with = hematoxylin=20 for 10 seconds.

Evaluation of EphA7 expression

The membranous and cytoplasmic expression of EphA7 on tumor = cells was=20 assessed at a =C3=97400 magnification. The assigned score first = reflects the=20 staining intensity A (0, negative; 1, weak; 2, moderate; 3, high) = and=20 second the percentage of positive cells B (0, no positive cells; = 1,=20 <25% positive cells; 2, 25 to 50% positive cells; 3, >50% = positive=20 cells). An overall score of 3 is defined as positive staining. The = scoring=20 was performed separately by two independent observers who were = blinded to=20 the clinical data. Any discrepancies were resolved on the = conference=20 microscope.

Evaluation of MVD at "hot spot" of tumor angiogenesis

Tumor angiogenesis can be reflected by MVD in the most = vascularised=20 areas of tumor tissue. MVD, as highlighted by factor VIII-related = antigen=20 immunostaining, was assessed without knowledge of the patient's = clinical=20 outcome, as described by Weidner et al [32]. = Briefly,=20 each slide was scanned at low magnification (=C3=97100) to = identify four areas=20 with the highest density of microvessel (hot-spots). Each hot-spot = was=20 then evaluated at high power magnification (=C3=97200) for the = number of=20 stained microvessels per field in a 0.7386 mm²surface = area.=20 vWF-positive stained blood vessels with a complete lumen as well = as cell=20 clusters without lumina were considered as individual = microvessels. The=20 final microvessel score was the average of vessel counts from four = fields=20 assessed by a high power magnification field (=C3=97200).

Statistical analysis

Survival curves were estimated using the Kaplan-Meier method. = The=20 distributions of survival were compared using the log rank test. = The=20 chi-square test was employed to determine the association between = EphA7=20 expression intensity on tumor cells and MVD. A p-value < 0.05 = was=20 considered to be statistically significant. All statistical = analysis was=20 performed using SPSS software.

Results

Demographic factors

32 patients with histologically confirmed primary and recurrent = GBM,=20 WHO grade IV, were studied. The mean age at diagnosis was 54.3 = years=20 (range 31=E2=80=9371). No significant difference in age = distribution between male=20 (21 cases) and female (11 cases) was detected. All of the 32 = patients=20 showed a relapse between 1 and 22 months after surgery and = subsequently=20 died of the disease (median survival 15 months).

EphA7 immunoreactivity in GBM and normal brain tissues

In 22 of 32 patients, EphA7 immunoreactivity was observed on = the=20 prominent membrane and cytoplasm of tumor cells showing different=20 intensities of EphA7 protein. Representative photomicrographs = illustrating=20 specimens with negative and strong EphA7 expression in tumor cells = are=20 presented in Fig. 1A = and 1B,=20 respectively. EphA7 protein expression in glioma cells of 10 = normal brain=20 tissues analyzed was undetectable as it was shown in Fig. 1E. = Of=20 the 32 GBM analyzed, strong expression of EphA7 (staining = intensities of=20 from 6 to 9) was observed in 14 cases (43.7%) of GBM. The staining = was=20 specific in both tumor and endothelial cells, with minimal = staining of=20 surrounding connective tissues.

Figure 1. = Immunohistochemical=20 demonstration of EphA7 protein expression and blood vessels in=20 GBM. Representative examples of GBM showing negative = staining in=20 tumor cells (A) or strong membranous and cytoplasmic staining in = both=20 tumor cells and endothelial cells (B). MVD in GBM by = immunohistochemical=20 staining for vWF, microvessels are represented by brown clusters, = which=20 stand out sharply from other tissues. Low tumor vascularity (C) in = GBM=20 with low expression of EphA7 as shown A. In contrast, microvessel = density=20 was relatively high (D) in GBM with high expression of EphA7 as = shown B.=20 Negative staining in normal brain tissue (E). Original = magnification, =C3=97400=20 (A, B, E) and =C3=97200 (C, D).

EphA7 associated with MVD in GBM

All tumors were stained with vWF and microvessels were counted = as a=20 measure of tumor's angiogenic activity. The median MVD of 30 = vessels was=20 treated as a cutpoint. High MVD was noted in areas where = overexpression of=20 EphA7 was marked (Table 1).=20 Representative examples of low or high MVD were showed in Fig 1C = and 1D,=20 respectively. There was a statistically significant correlation = between=20 expression of EphA7 and MVD in the tumors (P =3D 0.004, Table 1).

Tab= le=20 1. Relationship between EphA7 expression and = intratumor=20 microvessel density in 32 primary and recurrent = GBM.

EphA7 immunoreactivity predicted overall survival but not=20 recurrence-free survival

The median survival of patients with positive EphA7 expression = was=20 reduced in comparison with patients with negative EphA7 = expression. EphA7=20 protein expression showed an inverse correlation with the overall = survival=20 (p =3D 0.02, Fig. 2A).=20 However, the level of EphA7 expression did not emerge as a = prognostic=20 factor for recurrence-free survival of GBM patients (p =3D 0.51, = Fig. 2B).=20 There was no correlation between MVD and overall survival or=20 recurrence-free survival (p > 0.05, data not shown). We further = explored the prognostic relationship using EphA7 in combination = with MVD.=20 The study cohort could be divided into 3 groups based on = expression for=20 EphA7 and MVD combination: EphA7(+)/high MVD (n =3D 12), = EphA7(+)/low MVD (n=20 =3D 10), EphA7(-)/high MVD (n =3D 0), EphA7(-)/low MVD (n =3D 10). = As shown in=20 Fig. 2C, = high=20 density of MVD as well as EphA7 expression predicted for the = disease=20 outcome more accurately than Eph variable alone (p =3D 0.01).

Figure 2. Kaplan-Meier = curve for=20 overall survival and recurrence-free survival in 32 patients based = on=20 EphA7 expression and MVD index. (A) Increased EphA7 = expression=20 was significantly associated with dead of disease (P =3D = 0.02 by=20 log-rank test), when positive EphA7 (score =3D 4=E2=80=939) = expressing tumors were=20 plotted against negative EphA7 expressing tumors (score =3D = 0=E2=80=933). (B) EphA7=20 expression revealed no significance for recurrence-free survival. = (C)=20 EphA7 expression in combination with high MVD showed an inverse = outcome (p=20 =3D 0.01).

Clinical features associated with EphA7 or MVD

Statistical correlation was detected between the expression = levels of=20 EphA7 or MVD and clinical pathological parameters such as age, = gender and=20 tumor status (Table 2). A=20 statistically significant correlation between higher MVD and tumor = recurrence was observed (p =3D 0.003). In addition, positive EphA7 = expression was associated with increased age of patients (>55 = years, p=20 =3D 0.003).

Tab= le=20 2. Relationship between EphA7 expression or = microvessel count=20 and clinicopathological features of 32 patients with=20 GBM.

Discussion

There is currently an urgent need for development of = alternative,=20 effective diagnostic and therapeutic approaches to GBM. The = survival of=20 patients with GBM may depend on the identification of novel = targets. EphA2=20 receptor has already been recognized as a potential molecular = marker and=20 target in GBM for the development of novel biological therapeutic = agents=20 [22,23,33]. = Whereas=20 several studies in recent years have clearly indicated that = altered=20 expression of Eph receptors and ephrin ligands is associated with=20 increased potential for tumor growth, angiogenesis, metastasis and = adverse=20 outcome [34-42], few = studies have addressed the role of EphA7 in tumor pathogenicity. = By=20 employing immunohistochemical techniques we have found that EphA7 = protein=20 is predictive for the outcome of patients with GBM, independent of = MVD=20 expression. The data in the present study revealed for the first = time a=20 strong correlation between EphA7 overexpression and patient = survival.

Hafner C, et al. found that EphA7 is highly expressed in kidney = vasculature [43]. = The mRNA=20 of EphA7 is strongly upregulated in hepatocellular carcinoma as = compared=20 with healthy liver tissue and is downregulated in colon = carcinomas. EphA7=20 is also transcriptionally activated in lung cancer [44].=20 Furthermore, overexpression of EphA7 protein is frequently found = in=20 younger patients and in patients with advanced gastric carcinoma = [45]. = EphA7=20 expression is frequently silenced in human colorectal carcinoma by = aberrant promoter methylation [46]. = EphA7 is=20 located on 6q16.1, a region in close proximity to the chromosome 6 = breakpoint found in various types of cancer [47]. = Although=20 our findings are not consistent with Wang et al, who found a = significant=20 downregulation of EphA7 in colorectal carcinoma [46], = they are=20 in line with previous reports reporting a tumor promoter role in = lung=20 cancer and hepatocellular carcinoma [43,44],=20 implicating tumor type-specific function for different Eph family = members.=20 Eph receptors expressed in different cell types may have opposite = effects=20 due to cell-type specific intracellular signaling pathway [4]. = Indeed,=20 EphB4 receptor has been identified as a tumor suppressor in breast = cancer,=20 through activation of Abl-Crk antioncogenic pathway [48], = while the=20 same receptor presented a tumorigenic potential in mesothelioma, = favoring=20 uncontrolled cell growth, migration, and tumor progression [49]. = Moreover,=20 membrane-bound ephrins trigger Eph receptor phosphorylation, while = soluble=20 forms can bind to Eph receptor, but do not trigger receptor = activation [50]. = Murine and=20 human peripheral lymphocytes secrete a truncated form of EphA7 [51]. = Truncated=20 Eph receptors retaining their ligand-binding capacity have been = shown to=20 block activation of the full-length receptor [52]. = Promoter=20 hypermethylation and silencing of EphA7 in mature B-cell lymphomas = may=20 serve to eliminate the inhibitory activity of secreted EphA7 on=20 tumor-promoting EphA7 receptor signaling, thus enhancing tumor = cell spread=20 and recruitment of accessory cells able to promote tumor growth [51]. = A recent=20 study on signaling pathways involved in EphA7 RTK reported that = direct=20 EphA7 knockdown can result in attenuation of ERK1/2 = phosphorylation and=20 induce apoptosis of leukemia cells, suggesting the impact of EphA7 = on the=20 growth of tumor cells [45]. = It is of=20 interest that positive EphA7 expression was closely associated = with=20 increased age of patients (>55 years). Whether this is a random = finding=20 or not deserves further investigation.

The unfavorable prognostic influence of EphA7 in GBM could be=20 attributed to the well-recognised role of Eph RTKs in tumor = angiogenesis.=20 Indeed, in this study a statistically significant correlation = between=20 expression of EphA7 and MVD was noted in GBM specimens. Another = important=20 observation was EphA7 overexpression in both vasculature as well = as tumor=20 cells. The process of angiogenesis plays a central role in tumor = growth=20 and in the development of distant metastases by facilitating the = entry of=20 cells into circulation [52-55]. A = vast=20 biochemical and genetic evidence has implicated the critical role = of=20 Eph/ephrin signaling in angiogenesis, despite of VEGFR2 and Tie2 = receptors=20 long been recognized as key players in this process [53,54].=20 Angiogenetic activity can be measured histologically by MVD, which = has=20 been shown to be an independent prognostic parameter in various=20 malignancies including gliomas [56-58].=20 However, other studies on angiogenesis of glioblastomas suggested = the=20 limited usage of MVD as prognostic parameter due to the complexity = of the=20 microvascular network in GBM [59,60]. = Although=20 no correlation between MVD and overall survival or recurrence-free = survival was found in our study, we observed a statistically = significant=20 correlation between lower MVD and tumor recurrence. Further = prospective=20 studies with large numbers of patients are, however, needed to = fully=20 clarify the clinical implications of MVD in GBM recurrence.

Conclusion

Taken together, our data illustrated that EphA7 could be a = potential=20 candidate as a prognostic tumor marker and a new targeted = therapeutic=20 assessment in primary and recurrent GBM. Based on our findings, = there=20 might be a possible relationship between EphA7 and tumor=20 neovascularization. Recent data demonstrating inhibition of = angiogenesis=20 through EphA receptor blockade in two different animal tumor = models are=20 consistent with our observation [52]. = Additional=20 experimental work is necessary to unveil the biologic pathway = linking=20 Eph/ephrins with tumor growth in cancer cells and tumor-associated = vessels=20 of GBM and further studies are needed before EphA7 becomes = established as=20 an important prognostic and predictive tool in GBM. Ultimately, = specific=20 EphA7 inhibitors may prove to be of therapeutic value.

Abbreviations

GBM, glioblastoma multiforme; RTKs, Receptor tyrosine kinases; = MVD,=20 microvascular density; vWF, von Willebrand factor.

Competing interests

The author(s) declare that they have no competing = interests.

Authors' contributions

LFW, EF and JJ carried out the Immunohistochemical studies. AY = and FR=20 participated in the design of the study and performed the = statistical=20 analysis. AP and HXA participated the evaluation of analysed = parameters=20 and tumor pathological characteristics. REC conceived of the study = and=20 participated in the design and coordination as well as helped to = draft the=20 manuscript. All authors read and approved the final = manuscript.

Acknowledgements

We are very grateful to our technicians, Mrs. B. Kleb, H. = Gei=C3=9Fel and R.=20 Wassmuth for their expert technical assistance.

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