Cilengitide in bevacizumab-refractory high-grade glioma: two case reports and critical review of the literature
Giuseppe Lombardia, Fable Zustovicha, Patrizia Farinaa, Valentina Poloa, Miriam Farinaa, Alessandro Della Puppad, Roberta Bertorelleb,
Marina Paola Gardimane, Franco Bertic and Vittorina Zagonela

High-grade gliomas (HGG) are aggressive and highly vascularized brain tumours. Despite multimodality therapy including surgery, radiation therapy and in many cases temozolomide chemotherapy, the prognosis is dismal. Salvage therapies following progression after radiation therapy and chemotherapy have historically yielded disappointing results. Bevacizumab is an interesting antiangiogenic drug used as a second-line treatment
but although most patients benefit, essentially all patients ultimately progress. Moreover, some clinical studies have documented low activity of a second attempt at vascular endothelial growth factor pathway inhibition after failure of a first. The use of another drug with a different angiogenic pathway inhibition may probably result in a higher activity. Here, we describe, to our knowledge for the first time, the activity and safety of cilengitide, an agent with a different
antiangiogenic and anti-invasive activity, administered in two bevacizumab-refractory patients with HGG. In addition, we present a rapid review of the activity of cilengitide in HGG. Anti-Cancer Drugs 23:749–753 cti 2012 Wolters Kluwer Health | Lippincott Williams & Wilkins.

Anti-Cancer Drugs 2012, 23:749–753

Keywords: angiogenesis, bevacizumab, cilengitide, high-grade glioma, recurrent glioma
aMedical Oncology 1 Unit, Departments of bMolecular Immunology and Oncology, cRadiotherapy and Nuclear Medicine, Venetian Oncology Institute – IRCCS, Departments of dNeurosurgery and ePathology, Neurological Sciences, Padua Hospital, University of Padua, Padua, Italy
Correspondence to Giuseppe Lombardi, MD, Medical Oncology 1 Unit, Venetian Oncology Institute – IRCCS, via Gattamelata, Padua 64 35128, Italy
Tel: + 39 340 059 3278; fax: + 39 049 821 5904; e-mail: [email protected]

Received 13 January 2012 Revised form accepted 1 February 2012

Introduction
High-grade gliomas (HGG), including anaplastic astro- cytoma, anaplastic oligodendroglioma, mixed anaplastic oligoastrocytoma and glioblastoma, are highly vascularized brain tumours and are therefore attractive targets for anti- angiogenic therapies [1]. Vascular endothelial growth factor (VEGF) is an important regulator of angiogenesis and invasion and is highly expressed within brain tum- ours. Bevacizumab, an anti-VEGF antibody, was approved by the US Food and Drug Administration in 2009 for patients with recurrent glioblastomas who did not show a response to previous temozolomide and radiation therapy. Moreover, a recent clinical trial showed encouraging evidence of bevacizumab activity as well as acceptable safety among patients with recurrent grade III malignant glioma [2]. Large phase III studies evaluating bevacizu- mab-containing regimens in patients with newly diag- nosed glioblastoma have recently completed enrolling patients (AVAGLIO and Radiation Therapy Oncology Group-0825 studies).
Despite the benefits of antiangiogenic drugs in the treat- ment of HGG, tumour progression is inevitable. Various prospective and retrospective studies have demonstrated that continuation of bevacizumab and addition of or switching to another cytotoxic agent in patients who progress on previous bevacizumab-based treatment has a low activity, with a median time to progression from
1to 2.6 months [3]. Replacement of bevacizumab with

another antiangiogenic drug with a different molecular target may potentially increase efficacy.
A more recent agent of interest with antiangiogenic and anti-invasive activity is cilengitide, a specific inhibitor of avb3 and avb5 integrins [4], with an interesting activity in HGG [5].
We describe, for the first time, to our knowledge, two cases of patients with HGG treated with cilengitide after progression on previous bevacizumab-based treatment.

Case reports
First patient
The first patient was a 25-year-old woman with an anaplastic astrocytoma, with an unmethylated O6-methyl- guanine-DNA methyltransferase (MGMT) gene promoter and wild-type isocitrate dehydrogenase (IDH)-1 and 2 genes, infiltrating the corpus callosum. After a biopsy, she was treated with radiotherapy (60 Gy in 30 fractions) plus temozolomide (75 mg/m2/day for 42 days), followed by 12 cycles of temozolomide (150–200 mg/m2/day for 5 days every 28 days). After 6 months of temozolomide treat- ment, there was a progression in disease assessed on the basis of the Macdonald criteria [6]. Thus, the patient was started on bevacizumab (10 mg/kg) plus irinotecan (125 mg/m2) every 2 weeks, achieving stable disease after
2months of treatment. After 12 cycles of therapy, a gadolinium-brain MRI showed new progression. The

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Fig. 1

Axial T1-weighted, gadolinium-enhanced MRI of the brain before (a) and after (b) cilengitide treatment.

patient had an Eastern Cooperative Oncology Group performance status of 2. Two weeks after the previous treatment and after obtaining written, informed consent and approval from the Independent Ethics Committee of the Istituto Oncologico, she received cilengitide 2000 mg, administered intravenously twice weekly. After 2 months of therapy, the patient was subjected to a complete physical examination, combined with brain MRI findings, which included evaluation of both enhancing and T2/fluid- attenuated inversion recovery sequences according to the Radiologic Assessment in Neuro-Oncology (RANO) criteria [7]. Figure 1 shows the axial T1-weighted, gadolinium-enhanced MRI of the brain before (Fig. 1a) and after (Fig. 1b) cilengitide treatment; Fig. 2 shows the axial fluid-attenuated inversion recovery brain MRI before (Fig. 2a) and after (Fig. 2b) cilengitide treatment. According to the RANO criteria, on the basis of clinical status, corticosteroid dose and brain MRI, the patients had stable disease; moreover, no significant toxicity was experienced during the treatment. However, because of logistical problems, the patient decided to discontinue the treatment and, the following month, her clinical condition worsened. Four months after starting cilengi- tide, she died.

Second patient
The second patient was a 46-year-old man with a frontal anaplastic astrocytoma with a methylated MGMT gene promoter, IDH-1 mutation in codon 132 (R132H) and wild-type IDH-2. He underwent a gross total resection, followed by concomitant radiotherapy and temozolomide, and then temozolomide alone according to the Stupp regimen [8]. After six cycles of chemotherapy, he was diagnosed with a recurrence of disease. Thus, he was started on bevacizumab (10 mg/kg every 2 weeks) alone for 4 months until new disease progression was found on the basis of the RANO criteria. After 2 weeks, he was
started on cilengitide 2000 mg twice weekly, with no significant toxicity; his Eastern Cooperative Oncology Group performance status was 2. After 2 months of treatment, a gadolinium-brain MRI and clinical examina- tion demonstrated stable disease according to the RANO criteria. Because of logistical problems, treatment was discontinued in this patient and the following month he died.

Discussion
We describe, to our knowledge for the first time, the activity and safety of cilengitide, administered in two bevacizumab-refractory patients with HGG; both pa- tients were treated with cilengitide as third-line che- motherapy. We did not find any patients on published cilengitide trials who had received bevacizumab. This could either be because these patients were excluded from the protocol or because this information was not provided by the researchers.
Integrins are heterodimeric transmembrane cell surface receptors modulating the extracellular matrix and are an essential component of angiogenesis, tumour cell migra- tion, proliferation and survival. They are highly expressed on glioma tumour cells and tumour vasculature cells [4]. Moreover, specific integrin cooperation with particular growth factor receptors may confer responsiveness to specific angiogenic growth factors. In fact, avb5 and VEGF receptor-2 may promote VEGF-induced angiogen- esis. Thus, by targeting the angiogenic endothelial and tumour cell integrins, cilengitide may promote tumour cell apoptosis and normalize tumour vasculature [4]. It has been demonstrated that during bevacizumab therapy, tumours may evade the inhibition of VEGF signalling by selection of tumour cells that overexpress one or more of the alternative proangiogenic factors or by alternative upregulation of additional proangiogenic pathways such as

Fig. 2

Axial fluid-attenuated inversion recovery (FLAIR) brain MRI before (a) and after (b) cilengitide treatment.

Table 1 Studies analysing the activity of cilengitide in high-grade glioma

References

Dosage

Disease setting

Study
Patients enroled
Efficacy
(responses/survival)

Main toxicity

Nabors et al. [11] 120–2400 mg/m2
twice a week
Recurrent malignant glioma GBM (37 patients)
AA (11 patients) AO (one patient)
Mixed AA (two patients)
Phase I
51 2 CR (4%)
3 PR (6%) 16 SD (31%)
OS 5.6 monthsa
Hyperglycaemia (4%) Hyponatraemia (4%)

Reardon et al. [12] 500 or 2000 mg
twice a week
Recurrent GBM
Phase II
81 6 PR (9%) TTP 8.1 weeksa
OS 9.9 monthsa
Lymphopenia (9%) Neutropenia (1%)

Stupp et al. [13] 500 mg twice
a week
First-line GBM
(in combination with RT and TMZ)
Phase I/IIa 52 NA TTP 8 monthsa
OS 16.1 monthsa
6-month PFS 69%
Lymphopenia (56%) Thrombocytopenia (14%)

Gilbert et al. [14] 2000 mg twice
a week
Recurrent GBM planned for
second surgery
Phase II
26
NA
TTP 8 weeksa
6-month PFS 12%
Lymphopenia (19%)

AA, anaplastic astrocytoma; AO, anaplastic oligodendroglioma; CR, complete response; GBM, glioblastoma multiforme; NA, not available; OS, overall survival; PFS, progression-free survival; PR, partial response; RT, radiotherapy; SD, stable disease; TMZ, temozolomide; TTP, time to progression.
aMedian value.

compensatory overexpression of both avb3 and avb5 integrins [9]. Moreover, inhibition of VEGF could activate or increase the affinity state of integrins [10]. In contrast, activation of integrins could in turn increase tumour cell secretion of VEGF, providing a feedback loop resulting in increased tumour growth [4]. Thus, com- bined or sequential inhibition of VEGF and integrins may lead to further enhancement of the antitumour effect, and experience with our patients demonstrates that this approach could be effective and safe.

Several studies have analysed cilengitide in the treatment of HGG (see Table 1). In particular, a previous phase I/II study [13], analysing the use of cilengitide in combina- tion with radiotherapy and temozolomide in the adjuvant treatment of glioblastoma, showed that patients with a methylated MGMT gene promoter benefit significantly
from adjuvant cilengitide treatment; however, Reardon et al. [12] did not report a response to salvage cilengitide as a function of MGMT expression. The benefit seen in patients with promoter methylation of the DNA-repair enzyme gene MGMT may possibly be correlated to temozolomide or to IDH status (prognostic markers), and not to cilengitide; thus, patients with methylated MGMT have a better prognosis but it cannot yet be used as a predictive biomarker of cilengitide benefit. In fact, our patients showed a similar response, although their MGMT promoter genes had different methylation status. In addition, our patients had anaplastic astrocytomas and the predictive value of MGMT on response and survival may be different from those in patients with glioblasto- mas [15,16]. However, cilengitide in combination with temozolomide-based radiochemotherapy is currently being explored in a phase III trial for newly diagnosed

glioblastoma with MGMT promoter methylation (CEN- TRIC) and in a randomized phase II trial for newly diagnosed glioblastoma with an unmethylated MGMT promoter (CORE).
We also performed IDH-1 and two gene analyses showing a different IDH-1 mutation status between the two patients. IDH-1 mutation is a major prognostic factor for survival in patients with HGG; Yan et al. [17] identified mutations of IDH in more than 70% of grade II and III astrocytomas and oligodendrogliomas and in secondary glioblastomas, demonstrating that patients with such tumours have a better outcome than those with wild- type IDH genes. However, to date, there are no data on the predictive value of the IDH-1 mutation in patients treated with antiangiogenic drugs; perhaps, in patients with IDH mutations, antiangiogenic drugs might have greater activity than that in patients with wild-type IDH. IDH mutations result in a reduced enzymatic activity towards the native substrate, isocitrate, and mutant IDH catalyses the formation of 2-hydroxyglutarate from a- ketoglutarate (aKG) [18]. aKG promotes the degradation of hypoxia-inducible factor (HIF)-1a; under normoxic conditions, the activation of the HIF pathway results in the induction of angiogenesis. It has been demonstrated that decreased IDH activity produces a reduction in aKG levels, which in turn can lead to stabilization of HIF-1a; thus, IDH-mutated HGG exhibit an increase in angiogen- esis compared with gliomas without IDH mutations [19]. However, in our cases, although the patients discontinued the treatment and cilengitide administration was third- line chemotherapy, no difference in response according to the RANO criteria emerged between the patients.

In addition, our patients developed rapid disease progression after stopping cilengitide therapy. This may indicate the importance of a prolonged and continuous administration of cilengitide. Reynolds et al. [20] recently reported that cilengitide may, under certain experimental conditions such as low plasma levels, promote rather than inhibit angiogenesis; perhaps discontinuation of cilengi- tide administration could lead to a decrease in plasma levels and concentrations in the tumour, promoting angiogenesis. Gilbert et al. [14], in a randomized trial evaluating two doses of either 500 or 2000 mg cilengitide before tumour resection in recurrent disease, showed that cilengitide was detected in all tumour specimens, with higher levels in the group receiving 2000 mg dosing.

However, in all previous clinical trials, cilengitide was well tolerated; the most frequent adverse events were lymphopenia and thrombocytopenia, with huge differ- ences among the trials (see Table 1). In our cases, both patients refused to continue cilengitide treatment due to logistical problems and not due to its toxicity; in fact, the continuous administration of intravenous cilengitide twice per week can negatively impact quality of life and lead patients to discontinue the treatment.

Regarding sequential targeted therapy, recently, Goldlust et al. [21] showed an interesting activity of bevacizumab in patients with HGG treated with VEGF receptor targeting tyrosine kinase inhibitor, such as cediranib and sunitinib. These data and our case reports demonstrate that failure to respond to an ineffective antiangiogenic therapy may not preclude response to another targeted drug with a different angiogenic pathway inhibition.
In conclusion, although we treated only two patients, who discontinued treatment 2 months after starting therapy, cilengitide might be an interesting and safe treatment in patients with HGG who have a relapse after previous bevacizumab-based therapy. The probable synergistic activity between cilengitide and bevacizumab against HGG should also be evaluated in clinical trials analysing the combined inhibition of integrins and VEGF.

Acknowledgements
Conflicts of interest
There are no conflicts of interest.

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