**3. Angiogenesis**

#### **3.1. History**

The theory that tumor growth is dependent on angiogenesis and that anti-angiogenic therapy may be a potential cancer treatment was first proposed by Dr. Folkman in the 1970s (Folkman 1972). Since that time, understanding the mechanism of action of angiogenesis and developing targeted therapies have been a high priority.

#### **3.2. Summary of angiogenesis**

**2. Overview and significance**

178 Tumors of the Central Nervous System – Primary and Secondary

in this chapter (Omuro and DeAngelis 2013).

approximately 2 to 5 years (Wen and Kesari 2008).

targeted therapies have been a high priority.

**3. Angiogenesis**

**3.1. History**

**2.2. Significance of malignant gliomas**

CNS neoplasms are diverse and demonstrate a great deal of variability in terms of clinical presentation, aggressiveness, and response to therapy, with distinctions in histology and cellular and molecular composition being primarily responsible for these variations (Brat and Mapstone 2003; Omuro and DeAngelis 2013). Gliomas are the most frequent primary brain tumors in adults and, of this group, anaplastic astrocytomas and glioblastoma (GBM) are the two highest-grade astrocytic neoplasms (Brat and Mapstone 2003; Ricard, Idbaih et al. 2012). The World Health Organization (WHO) system classifies astrocytomas into four grades. These histological grades are defined by increasing degrees of undifferentiation, anaplasia, and aggressiveness (Louis, Ohgaki et al. 2007; Omuro and DeAngelis 2013). Grade I and II tumors, the lower grade tumors, are well-differentiated with limited cell density. The characteristic features of grade III astrocytomas (anaplastic) are increased vessel and cell density, cellular atypias, and high mitotic activity. Grade IV astrocytoma (GBM) is characterized by vascular proliferation or necrosis (Westphal and Lamszus 2011; Omuro and DeAngelis 2013). Glioblas‐ toma and other malignant gliomas are highly infiltrative tumors. Of note, there is also a WHO grading system for oligodendrogliomas and oligoastrocytomas, but they will not be discussed

The annual incidence of malignant glioma is 5.26 per 100 thousand and this group accounts for approximately 80% of the total number of new cases of malignant primary brain tumors diagnosed in the United States each year (Omuro and DeAngelis 2013). The overall incidence of gliomas is highest among Caucasians, as compared to other ethnic groups, and is higher among males as compared to females (7.2 versus 5.0 per 100,000 persons-years) (Peak and Levin 2010). Malignant gliomas can occur in any age group; however, the incidence increases in the fifth decade of life and peaks at about 65 years of age (Brat and Mapstone 2003). GBM is the most aggressive glioma. Stupp and colleagues reported that 27.2 and 9.8 percent of GBM patients treated by concomitant and adjuvant Temozolamide and radiotherapy remained alive at 2 years and 5 years, respectively (Stupp, Mason et al. 2005; Stupp, Hegi et al. 2009). For patients diagnosed with anaplastic astrocytoma, the median survival time is higher at

The theory that tumor growth is dependent on angiogenesis and that anti-angiogenic therapy may be a potential cancer treatment was first proposed by Dr. Folkman in the 1970s (Folkman 1972). Since that time, understanding the mechanism of action of angiogenesis and developing

**2.1. Classification of gliomas**

Angiogenesis is the process by which the vascular system is formed through growth of new capillaries from pre-existing vessels (Plate, Scholz et al. 2012). Angiogenesis plays a critical role in key physiologic and formative processes such as embryogenesis, regeneration, and wound healing. Angiogenesis is also involved in various pathologic processes including age-related macular degeneration, rheumatoid arthritis, and tumor growth and development (Wang, Fei et al. 2004).

The process of angiogenesis can be briefly summarized as follows. First, there is vasodilation, in response to nitric oxide, and increased permeability of the existing vessels. This is followed by degradation of the existing vessel's basement membrane. Next, endothelial precursor cells migrate to the area and begin to proliferate and mature into capillaries via a balance of both growth and inhibition. The final steps involve recruitment of vascular smooth muscle cells and pericytes that form a new network of mature vessels (Shinkaruk, Bayle et al. 2003).

#### **3.3. Molecular signals of angiogenesis**

Although there are numerous factors and signals that contribute to angiogenesis, the chemical signal that seems to play the most critical role in the process is Vascular Endothelial Growth Factor (VEGF). VEGF is a pro-angiogenic growth factor, which is secreted by many cells, including mesenchymal, stromal, and especially tumor cells. VEGF induces the migration of the endothelial precursor cells to sites of angiogenesis and is responsible for their proliferation and differentiation. The VEGF gene is located on chromosome 6p12 and the gene family is composed of five members, namely VEGF-A, VEGF-B, VEGF-C, VEGF-D, and placentalderived growth factor (PlGF). Of these, VEGF-A, B, and PIGF are involved in the development of the vascular system and VEGF-C and D are involved in the development of the lymphatic system (Ahluwalia and Gladson 2010). VEGF primarily signals through its receptor VEGFR2 which is a tyrosine kinase receptor that is expressed by many cells, including endothelial cells, endothelial cell precursors, and tumor cells (Jain, di Tomaso et al. 2007). Other chemical signals that play an important role in angiogenesis are fibroblast growth factor, hepatocyte growth factor (HGF), tumor necrosis factor-alpha (TNF-α), transforming growth factor-beta (TGF-β), angiopoietins, and platelet derived growth factor (PDGF). Their various roles include involve‐ ment in extracellular matrix degradation, endothelial proliferation and migration, and neovessel stabilization and maturation (Martin and Jiang 2010; Ucuzian, Gassman et al. 2010).

#### **3.4. Angiogenesis in tumors**

Seven different cellular mechanisms appear to contribute to tumor angiogenesis: (1) classical sprouting angiogenesis, (2) vascular co-option, (3) myeloid cell-driven angiogenesis, (4) vessel intussusception, (5) vasculogenic mimicry, (6) bone marrow derived vasculogenesis, and (7) cancer stem-like cell derived vasculogenesis (Carmeliet and Jain 2011; Plate, Scholz et al. 2012). Of the above-listed mechanisms, the first three seem to have a clear role in glioma vascularization, as supported by pre-clinical tumor models (Plate, Scholz et al. 2012).

**1.** *Classical sprouting angiogenesis*. This is believed to be the primary modulator of neovascu‐ larization of the brain during development and in pathological conditions (Plate, Breier et al. 1994; Risau 1997; Kurz, Korn et al. 2001; Plate, Scholz et al. 2012). In this model, a vascular sprout is led by tip cells toward an angiogenic stimulus that is produced by tumor cells. This sprout then elongates via dividing stalk cells. The newly formed vessel undergoes remodeling to create a vascular lumen that allows blood flow (Plate, Scholz et al. 2012). There is evidence to support that both tip and stalk cell phenotypes co-exist in the glioblastoma vasculature (Plate, Breier et al. 1994; Broholm and Laursen 2004; Dieterich, Mellberg et al. 2012; Plate, Scholz et al. 2012)

bind to its target receptors on endothelial cells. This neutralization has been shown to have

Anti-Angiogenesis, Gene Therapy, and Immunotherapy in Malignant Gliomas

http://dx.doi.org/10.5772/58320

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Recently, two phase III clinical trials investigating Bevacizumab as a first-line treatment for newly diagnosed GBM tumors were completed. Unfortunately, both trials were consistent in showing no statistically-significant prolongation of overall survival time (OS) but there was a slight improvement in progression-free survival time (PFS). The two trials had a similar design, namely double-blinded prospective trials where newly diagnosed GBM patients were randomized to either standard of care with Bevacizumab or with placebo; the standard of care consisted of radiation therapy with adjuvant and concomitant Temozolomide. A total of 637 and 921 adult participants were randomized in the Radiation Therapy Oncology Group (RTOG) and AVAglio trials, respectively. The median OS was 16.1 vs. 15.7 months, in the RTOG trial (*p* = 0.11). The median PFS was longer in patients who received Bevacizumab, 7.3 vs. 10.7 months (*p* = 0.004) and 6.2 vs. 10.6 months (p<0.0001) in the RTOG and Avaglio trials, respectively. In addition, the results also showed a higher incidence of adverse reactions in the Bevacizumab arm, including neutropenia, hypertension, and deep vein thromboembolism and pulmonary emboli (Gilbert, Dignam et al. 2013). The AVAglio trial noted delayed time to definitive deterioration in terms of health-related quality of life (p<0.0001) and Karnofsky Performance Scale, and increased time to corticosteroid initiation (HR 0.71, 95% CI 0.57-0.88; median 12.3 vs. 3.7 months) (Henriksson, Bottomley et al. 2013). These results are discouraging and do not justify the use of Bevacizumab in a GBM patient who has had a reasonable surgical

The data support the idea that Bevacizumab may be reserved until the time of recurrence as several prospective phase II clinical trials have shown prolongation of the 6-month PFS rates ranging from 25 to 42.6 percent and median OS times from 6.5 to 9.2 months. However, a significant limitation of these trials is that the comparison was made to historical controls

VEGF-Trap (drug name Aflibercept) is a recombinant fusion protein that acts as a decoy receptor for VEGF, thereby blocking its interaction with its normal receptors and interrupting the VEGF signaling pathway (Holash, Davis et al. 2002). VEGF-Trap was developed by incorporating domains of both VEGF receptor 1 and VEGF receptor 2 fused to the constant region of human immunoglobulin G1. VEGF Trap has a high affinity for all isoforms of VEGF-A, as well as for PlGF, another pro-angiogenic agent that primarily acts on VEGF receptor 1 (Holash, Davis et al. 2002; Gomez-Manzano, Holash et al. 2008; de Groot, Lamborn et al. 2011). Preclinical studies demonstrated efficacy of VEGF-trap in glioma animal models (Haapa-Paananen, Chen et al. 2013). de Groot et al. conducted a Phase II study of Aflibercept in recurrent malignant glioma; unfortunately, their results revealed that Aflibercept had minimal

(Friedman, Prados et al. 2009; Kreisl, Kim et al. 2009; Raizer, Grimm et al. 2010).

activity as a single-agent against recurrent GBM (de Groot, Lamborn et al. 2011).

efficacy not only in *in vitro* studies, but also in *in vivo* ones.

resection.

*4.1.2. VEGF-trap*


The role of the remaining four mechanisms in glioma angiogenesis is not yet fully understood. Briefly, *vessel intussusception* is the process by which a new vessel is formed by internal division of the pre-existing capillary plexus without sprouting through a series of steps that include vascular invagination, intra-luminar pillar formation and remodeling, and splitting (Djonov, Schmid et al. 2000; Plate, Scholz et al. 2012). *Vasculogenic mimicry* refers to the process by which cancer cells form *de novo* vasculature as a result of their high plasticity (Plate, Scholz et al. 2012; Seftor, Hess et al. 2012). *Bone marrow-derived vasculogenesis* refers to the process by which circulating endothelial precursor cells are recruited to the tumor and are incorporated into the vessel wall (Plate, Scholz et al. 2012; Huang, Peng et al. 2013). *Cancer stem-like derived vasculo‐ genesis* is the process by which tumor-derived cells trans-differentiate into endothelial cells (Ricci-Vitiani, Pallini et al. 2010; Plate, Scholz et al. 2012). It is not the goal of this chapter to study these mechanisms in detail, but instead to provide an overview of angiogenesis in glioma and discuss key molecules involved and possible therapeutic options that target them.
