**3. Why interfering with HER1/EGFR or EGFRvIII-mediated signaling?**

Given the poor therapeutic efficacy of current treatment measures for glioblastoma, the need for different therapeutic strategies is evident. HER1/EGFR is the most frequently amplified gene in glioblastoma, and its overexpression was found in more than half of these tumors which renders HER1/EGFR an outstanding therapeutic target (Salomon et al., 1995). Experi‐ mental studies show that HER1/EGFR stimulates tumor growth, invasion and migration (Lund-Johansen et al., 1990). In addition, data from clinical studies suggest that HER1/EGFR amplification is related to decreased overall survival and worse prognosis in patients with glioblastoma (Lund-Johansen et al., 1990; Shinojima et al., 2003).

EGFRvIII represents the most common mutant form of HER1/EGFR and is characterized by constitutive TK activity independent of ligand-binding (Batra et al., 1995; Frederick et al., 2000). Analysis of the expression of HER1/EGFR and EGFRvIII in bioptic glioblastoma specimens suggests concurrent overexpression of both EGFRvIII and HER1/EGFR in most of the tumors (Biernat et al., 2004). Moreover, in an experimental study using a murine model of human glioma xenografts, EGFRvIII expression was found to be related to increased prolifer‐ ation, inhibition of apoptosis, and tumor formation (Nishikawa et al., 1994; Nagane et al., 1996). Other studies showed similar results and identified activation of the MAPK/ERK1/2 and PI3-K/Akt pathways as driving forces of cellular proliferation and tumor progression (Moscatello et al., 1998; Klingler-Hoffmann et al., 2001; Klingler-Hoffmann et al., 2003). In addition, in a murine orthotopic xenograft model of glioblastoma, administration of a mono‐ clonal antibody targeting EGFRvIII (mAb 806) was shown to cause a significant decrease of tumor growth, increase of apoptosis and prolongation of survival (Mishima et al., 2001).

The tumor-specific properties of EGFRvIII have also lead to the development of EGFRvIIItargeted vaccines in order to provoke an immunologic response against EGFRvIII-bearing glioblastoma cells. Potential antitumor efficacy of EGFRvIII-targeted vaccines had been shown by experimental studies. Immunization of mice with transfected allogenic 300.19/EGFRvIII cells was reported to induce a major histocompatibility complex class I-restricted response against EGFRvIII-bearing syngeneic B16-F10 melanoma or 560 astrocytoma cells that were implanted intracranially (Ashley et al., 1997). In addition, vaccinated animals were shown to have a significantly longer median survival upon intracranial tumor challenge when compared to controls. Similar findings were reported for mice that were vaccinated with PEP-3-KLH (rindopepimut, CDX-110, Celldex Therapeutics, Needham, MA, U.S.A.), a conjugate of a peptide comprising the tumor-specific mutated segment of EGFRvIII (PEP-3) and keyhole limpet hemocyanin (KLH) (Heimberger et al., 2003). In this study, C3H mice received vacci‐ nation with 100 µg of PEP-3-KLH 8, 6 and 2 weeks prior to intracerebral administration of K1735 murine melanoma cells that were transfected with a murine homologue of the human EGFRvIII, and additional vaccination 4 days after intracranial implantation of the tumor cells. A more than 173% longer survival time was shown for mice vaccinated with PEP-3-KLH when compared to mice receiving only KLH. Moreover, mice with already established intracranial tumors that were treated with a single dose of the PEP-3-KLH vaccine 4 days after adminis‐ tration of the transfected K1735 cells had a 26% increase of median survival. Based on these promising preclinical data, several clinical trials were conducted. In two phase II trials, vaccination with PEP-3-KLH was examined in patients with EGFRvIII-expressing newly diagnosed glioblastoma. In the ACTIVATE trial, 18 patients underwent gross-total tumor resection prior to radiotherapy and concurrent chemotherapy with temozolomide followed by vaccination with PEP-3-KLH bi-weekly for 3 doses and continued monthly until progres‐ sion (Sampson et al., 2010). The data were compared to a matched historical control group (n=17). The median progression-free survival and overall survival were 14.2 months and 26 months, respectively, versus 6.3 months and 15 months, respectively, in the control group. Notably, the patients who developed an EGFRvIII-specific antibody response had an overall survival of 47.7 months (n=6) compared to an overall survival of 22.2 months in patients lacking a specific antibody response (n=8). In the ACT II trial, 22 patients who met the same inclusion criteria as for the ACTIVATE trial received the same therapeutic regimen except for an additional treatment with temozolomide either at a dose of 200 mg/m2 for 5 days of a 28-day cycle or at a dose of 100 mg/m2 for 21 days of a 28-day cycle in conjunction with the vaccination therapy (Heimberger et al., 2009). Combination therapy of PEP-3-KLH and temozolomide was well tolerated, and a favorable median overall survival of 20.5 months was reported. An additional phase II study (ACT III) was conducted by Celldex Therapeutics. Sixty-five patients with newly diagnosed EGFRvIII-positive glioblastoma were enrolled in this single-arm multicenter study which was initially planned as a phase IIb/III randomized two-arm trial but had to be transformed into a single-arm design due to withdrawal of consent to participate in this study by 14 of the 16 patients that were randomized to the control group. In this study, a median overall survival of 21.8 months was reported which encouraged Celldex to launch two more studies: ACT IV, a randomized controlled phase III study in patients with newly diagnosed EGFRvIII-positive glioblastoma and ReACT, a phase II study in patients with EGFRvIII-positive recurrent glioblastoma. The final results of these studies are pending. However, what needs to be taken into account is the fact that only a part of the glioblastomas

express EGFRvIII. For this subset of patients, however, vaccination with PEP-3-KLH might

Erlotinib in Glioblastoma – A Current Clinical Perspective

http://dx.doi.org/10.5772/58296

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HER1/EGFR TK inhibitors such as erlotinib compete with adenosine triposphate and reversi‐ bly bind to the intracellular catalytic TK domain of HER1/EGFR or EGFRvIII thus inhibiting autophosphorylation of the receptor as well as further downstream signaling (Halatsch et al., 2006). In preclinical studies, erlotinib was shown to exert a variety of relevant antineoplastic effects in the setting of glioblastoma. Lal *et al*. showed that exposure of transformed D54-MG glioblastoma cells (D54-EGFRvIII) to 20 µM of erlotinib resulted in significant downregulation of certain genes encoding pro-invasive proteins and in significant inhibition of the invasiveness of D54-EGFRvIII cells (Lal et al., 2002). In a different study, erlotinib was shown to significantly reduce cellular viability of six human glioblastoma-derived tumor-initiating cell lines when given at a concentration of 5 µM (Griffero et al., 2003). This effect was shown to be in con‐ cordance with decreased EGF-induced phosphorylation of HER1/EGFR and subsequent inhibition of the MAPK signaling pathway by reduced phosphorylation of ERK1/2. Moreover, Halatsch *et al*. showed that the extent of erlotinib-mediated inhibition of anchorage-independ‐ ent growth of glioblastoma-derived cell lines correlates inversely with the cellular capability to induce HER1/EGFR mRNA, emphasizing the important role of HER1/EGFR in the patho‐

Based on the positive findings reported by preclinical studies, much hope was set on the clinical application of erlotinib in glioblastoma patients. To date, several published studies have examined the effects of erlotinib on patients with recurrent or newly diagnosed glioblastoma. In phase I trials, erlotinib exhibited a reasonable safety profile and was generally well tolerated (Krishnan et al., 2006; Prados et al., 2006). In addition, EIAEDs were shown to accelerate drug metabolism of erlotinib which requires dose modification of erlotinib or a change in the antiepileptic drug regimen (Stupp et al., 2006). In terms of clinical efficacy, Raizer *et al*. examined the effects of erlotinib applied at a dose of 150 mg/d on 42 patients with recurrent glioblastoma and 43 patients with non-progressive glioblastoma following radiotherapy in a phase II trial (Raizer et al., 2010). For the patients with recurrent glioblastoma, median overall survival was reported as 6 months and median progression-free survival as only 2 months. Median overall survival and the 12-month overall survival were reported as 14 months and 57%, respectively, for the patients with non-progressive glioblastoma after radiotherapy. Thus, this study did not show a significant improvement of the clinical outcome attributable to the treatment with erlotinib in patients with recurrent glioblastoma or non-progressive glioblas‐ toma after radiotherapy. However, Yung *et al*. showed that median overall survival and 6 month progression-free survival of 48 patients with recurrent glioblastoma who were treated with erlotinib reached or exceeded historical values for patients receiving chemotherapy for recurrent glioblastoma (Yung et al., 2010). Notably, this study was discontinued due to an insufficient number of responses after a planned interim analysis, and a control group was not included. Van den Bent *et al*. showed in a randomized controlled phase II trial that only 11.4%

confer a significant clinical benefit.

**4. Erlotinib for the treatment of glioblastoma**

genesis of glioblastoma (Halatsch et al., 2004).

express EGFRvIII. For this subset of patients, however, vaccination with PEP-3-KLH might confer a significant clinical benefit.
