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[130] Rogers L, Morris H, Lupica K. Effect of cranial irradiation on seizure frequency in adults with low grade astrocytomas and medically intractable epilepsy. Neurology (1993) 43:

[131] Rossi GF, Scerrati M, Roselli R. Epileptogenic cerebral low-grade tumors: effect of interstitial stereotactic irradiation on seizures. Appl Neurophysiol (1985) 48: 127 – 132.

[132] Von Wiezer W. Die roentgentherapie der traumatischen epilepsie. Monatsschr Psy‐

[133] Chalifoux R, Elisevich K. Effect of ionizing radiation on partial seizures attributable to malignant cerebral tumors. Stereotact Funct Neurosurg (1996-97) 67: 169 – 182.

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[137] Cmelak A, Abou-Khalil B, Konrad PE, Duggin D, Maciunas RJ. Low dose stereotactic radiosurgery is inadequate for medically intractable mesial temporal epilepsy: a case

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Neurosurgery (2000) 46: 157 – 168.

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1599 – 1601.

**Chapter 12**

**Current Applications of 5-ALA in**

**Glioma Diagnostics and Therapy**

Lei Teng, Mitsutoshi Nakada, Yutaka Hayashi,

Additional information is available at the end of the chapter

Gliomas, the most common primary brain tumors, are characterized by rapid proliferation, marked infiltration, and poor prognosis and are known for their dismal outcomes (Iacob & Din‐ ca, 2009; Lefranc et al., 2006). The infiltrative nature of malignant glioma makes complete resec‐ tion difficult, as tumor margins are unclear. Recurrence of glioma takes place within approximately 2 cm of the margins of the resected cavity owing to its invasive character (Aydin et al., 2001; Wallner et al., 1989). The use of fluorescence to delineate tumor margins intraopera‐ tively has emerged as a safe and effective tool for increasing the extent of resection. Therefore, methods that easily detect tumor margins during surgery would be extremely beneficial. 5-ami‐ nolevulinic acid (5-ALA) fluorescence-guided glioma resection is a rapidly growing, novel ap‐ proach to improve the extent of tumor resection with broad applications in both preclinical and clinical settings (Stummer et al., 2000; Stummer et al., 1998b; Stummer et al., 1998c). Intraopera‐ tive tumor fluorescence provided by the chemical compound 5-ALA assists surgeons in identi‐ fying the true tumor margin during resection of glial neoplasms, consequently increasing the extent of the resection. 5-ALA is the most studied fluorescer and has been used in many clinical trials, including a multicenter phase III randomized controlled trial. Recent controlled Phase III clinical trials have demonstrated that this surgical method enables more complete resection of contrast-enhancing lesions than conventional microsurgery and improves progression-free

survival in patients with malignant glioma (Pichlmeier et al., 2008; Stummer et al., 2006).

Photodynamic therapy (PDT) is a treatment modality that takes advantage of the cytotoxic ef‐ fects induced by a photosensitizer and light in the presence of oxygen (Norum et al., 2009). This

and reproduction in any medium, provided the original work is properly cited.

© 2013 Teng et al.; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use,

© 2013 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution,

distribution, and reproduction in any medium, provided the original work is properly cited.

Takeshi Yoneyama, Shi-Guang Zhao and

Jun-Ichiro Hamada

**1. Introduction**

http://dx.doi.org/10.5772/52428

**Chapter 12**

## **Current Applications of 5-ALA in Glioma Diagnostics and Therapy**

Lei Teng, Mitsutoshi Nakada, Yutaka Hayashi, Takeshi Yoneyama, Shi-Guang Zhao and Jun-Ichiro Hamada

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/52428

### **1. Introduction**

Gliomas, the most common primary brain tumors, are characterized by rapid proliferation, marked infiltration, and poor prognosis and are known for their dismal outcomes (Iacob & Din‐ ca, 2009; Lefranc et al., 2006). The infiltrative nature of malignant glioma makes complete resec‐ tion difficult, as tumor margins are unclear. Recurrence of glioma takes place within approximately 2 cm of the margins of the resected cavity owing to its invasive character (Aydin et al., 2001; Wallner et al., 1989). The use of fluorescence to delineate tumor margins intraopera‐ tively has emerged as a safe and effective tool for increasing the extent of resection. Therefore, methods that easily detect tumor margins during surgery would be extremely beneficial. 5-ami‐ nolevulinic acid (5-ALA) fluorescence-guided glioma resection is a rapidly growing, novel ap‐ proach to improve the extent of tumor resection with broad applications in both preclinical and clinical settings (Stummer et al., 2000; Stummer et al., 1998b; Stummer et al., 1998c). Intraopera‐ tive tumor fluorescence provided by the chemical compound 5-ALA assists surgeons in identi‐ fying the true tumor margin during resection of glial neoplasms, consequently increasing the extent of the resection. 5-ALA is the most studied fluorescer and has been used in many clinical trials, including a multicenter phase III randomized controlled trial. Recent controlled Phase III clinical trials have demonstrated that this surgical method enables more complete resection of contrast-enhancing lesions than conventional microsurgery and improves progression-free survival in patients with malignant glioma (Pichlmeier et al., 2008; Stummer et al., 2006).

Photodynamic therapy (PDT) is a treatment modality that takes advantage of the cytotoxic ef‐ fects induced by a photosensitizer and light in the presence of oxygen (Norum et al., 2009). This

© 2013 Teng et al.; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2013 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

photodamage may be due to direct cytotoxicity, vascular damage, and inflammatory and im‐ munological responses (Castano et al., 2006). The relative importance of these pathways to the therapeutic effects depends on the tissue oxygenation, photosensitizer formulation, distribu‐ tion, and light dosimeter (Norum et al., 2009). 5-ALA-induced protoporphyrin IX (PpIX) can al‐ so be used for 5-ALA-PDT rather than photodynamic diagnosis (PDD) by exposing the field to either blue or red laser light; however, a red light at 632 nm would target a much larger tumor volume owing to better light penetration (Eljamel et al., 2008). Activation of PpIX by red light in combination with oxygen causes cell death by apoptosis and necrosis through the release of cy‐ totoxic singlet oxygen (Peng et al., 1997). This tool may aid in overcoming the hurdle of residual tumor cells after traditional neurosurgery. Application of 5-ALA-PDT has been emerging as a new field and is expected to create a major breakthrough in addressing several unsolved medi‐ cal issues, especially photokilling of residual neoplastic cells.

ported for conventional porphyrin derivatives (Stummer et al., 2006; Toda, 2008). When giv‐ en orally, 5-ALA has been shown to produce fluorescence in glial neoplasms both *in vitro* and *in vivo* (Blake & Curnow, 2010). In addition, the fluorescence of PpIX is itself cytotoxic and has the potential for use as an adjuvant photodynamic therapy for neoplastic tissue that cannot be safely resected (Sherman et al., 2011). Since the administered 5-ALA is excreted into the urine within 24 hours after oral administration and does not remain in the skin, the occurrence of photosensitivity can be adequately prevented by avoiding sun exposure for

Current Applications of 5–ALA in Glioma Diagnostics and Therapy

http://dx.doi.org/10.5772/52428

251

**Figure 1.** Schematic representation of diagnostics and PDT of 5-ALA for glioma cells. The figure illustrates 5-ALA is converted to PpIX in malignant gliomas via an oral-intake of exogenous 5-ALA. In the presence of an appropriate light source with the specific wavelength, PpIX fluorescence acts bimodal function; fluorescence diagnostic marker and 5- ALA-PDT. Red fluorescence acts as a discriminating marker to assist neurosurgeons to visualize the extent and margins of tumors; 5-ALA-PDT results in a series of irreversible photochemical and photobiological events that cause directly

5-ALA, the metabolic precursor of heme in the heme biosynthesis pathway, is not itself fluorescent, but is metabolized into endogenous fluorescent PpIX (Gossner et al., 1998; Inoue et al., 2007; Kennedy & Pottier, 1992; Loh et al., 1993; Peng et al., 1997). Heme biosynthesis consists of a series of enzyme-catalyzed steps, involving 5-aminolevulinate synthases 1 and 2,

approximately 24 hours after administration.

damage and killing glioma cells.

**3. 5–ALA biology in glioma**

Developing technologies such as fluorescence operation-systems to enhance endogenous fluorescence has fairly recently been shown to delineate brain tumor margins intraoperatively (Babu & Adamson, 2012). 5-ALA fluorescence-guided resection shows great promise for furthering our surgical abilities and will become the standard of care for patients diagnosed with malignant glioma in the foreseeable future. The following review analyzes the recent literature in an effort to describe how these modalities involving 5-ALA in glioma diagnostics and therapy can and should be used in the treatment of patients with glioma. This article reviews recent developments in the use of 5-ALA for simultaneous imaging and 5-ALAtriggered photodamage in glioma patients (Figure 1).

### **2. General knowledge of 5–ALA**

5-ALA (molecular weight, 167.6) is a natural biochemical precursor for heme synthesis in living mammalian cells (Eljamel et al., 2008; Peng et al., 1997). Each cell metabolizes 5-ALA along a set pathway toward heme production, inducing the synthesis of the endogenous flu‐ orescent molecule, PpIX, through metabolic conversion in the mitochondria (Bottomley & Muller-Eberhard, 1988). When PpIX emits peak fluorescence at 635 nm with a peak excita‐ tion wavelength at 405 nm, it is observed as a red light through a filter that allows this wavelength to pass. Clinical use of 5-ALA for PDT or PDD has also been reported in derma‐ tology, urology, neurosurgery, otorhinolaryngology, gynecology, and gastroenterology for various epithelia and cancerous tissues (D'Hallewin et al., 1998; Guyon et al., 2012; Loning et al., 2004; Piotrowski et al., 2004). In the USA, it has been used with the approval of the Food and Drug Administration (FDA) as a therapeutic drug for solar keratosis. Its use has not yet been approved by the pharmaceutical authority in Japan, and it is only used after obtaining approval from the ethics committee of the respective institutes. The tumor tissue concentra‐ tion of the fluorescent dye peaks at 2–6 hours after oral administration and disappears by 12 hours. Administration of antacid should be avoided at the time of oral administration be‐ cause the dye is easily decomposed in the presence of alkali, and use of 5-ALA is contraindi‐ cated in cases of porphyria, a genetic disease. PpIX biosynthesized from 5-ALA is a phototoxic substance, although the incidence of skin photosensitivity is lower than that re‐ ported for conventional porphyrin derivatives (Stummer et al., 2006; Toda, 2008). When giv‐ en orally, 5-ALA has been shown to produce fluorescence in glial neoplasms both *in vitro* and *in vivo* (Blake & Curnow, 2010). In addition, the fluorescence of PpIX is itself cytotoxic and has the potential for use as an adjuvant photodynamic therapy for neoplastic tissue that cannot be safely resected (Sherman et al., 2011). Since the administered 5-ALA is excreted into the urine within 24 hours after oral administration and does not remain in the skin, the occurrence of photosensitivity can be adequately prevented by avoiding sun exposure for approximately 24 hours after administration.

**Figure 1.** Schematic representation of diagnostics and PDT of 5-ALA for glioma cells. The figure illustrates 5-ALA is converted to PpIX in malignant gliomas via an oral-intake of exogenous 5-ALA. In the presence of an appropriate light source with the specific wavelength, PpIX fluorescence acts bimodal function; fluorescence diagnostic marker and 5- ALA-PDT. Red fluorescence acts as a discriminating marker to assist neurosurgeons to visualize the extent and margins of tumors; 5-ALA-PDT results in a series of irreversible photochemical and photobiological events that cause directly damage and killing glioma cells.

### **3. 5–ALA biology in glioma**

photodamage may be due to direct cytotoxicity, vascular damage, and inflammatory and im‐ munological responses (Castano et al., 2006). The relative importance of these pathways to the therapeutic effects depends on the tissue oxygenation, photosensitizer formulation, distribu‐ tion, and light dosimeter (Norum et al., 2009). 5-ALA-induced protoporphyrin IX (PpIX) can al‐ so be used for 5-ALA-PDT rather than photodynamic diagnosis (PDD) by exposing the field to either blue or red laser light; however, a red light at 632 nm would target a much larger tumor volume owing to better light penetration (Eljamel et al., 2008). Activation of PpIX by red light in combination with oxygen causes cell death by apoptosis and necrosis through the release of cy‐ totoxic singlet oxygen (Peng et al., 1997). This tool may aid in overcoming the hurdle of residual tumor cells after traditional neurosurgery. Application of 5-ALA-PDT has been emerging as a new field and is expected to create a major breakthrough in addressing several unsolved medi‐

Developing technologies such as fluorescence operation-systems to enhance endogenous fluorescence has fairly recently been shown to delineate brain tumor margins intraoperatively (Babu & Adamson, 2012). 5-ALA fluorescence-guided resection shows great promise for furthering our surgical abilities and will become the standard of care for patients diagnosed with malignant glioma in the foreseeable future. The following review analyzes the recent literature in an effort to describe how these modalities involving 5-ALA in glioma diagnostics and therapy can and should be used in the treatment of patients with glioma. This article reviews recent developments in the use of 5-ALA for simultaneous imaging and 5-ALA-

5-ALA (molecular weight, 167.6) is a natural biochemical precursor for heme synthesis in living mammalian cells (Eljamel et al., 2008; Peng et al., 1997). Each cell metabolizes 5-ALA along a set pathway toward heme production, inducing the synthesis of the endogenous flu‐ orescent molecule, PpIX, through metabolic conversion in the mitochondria (Bottomley & Muller-Eberhard, 1988). When PpIX emits peak fluorescence at 635 nm with a peak excita‐ tion wavelength at 405 nm, it is observed as a red light through a filter that allows this wavelength to pass. Clinical use of 5-ALA for PDT or PDD has also been reported in derma‐ tology, urology, neurosurgery, otorhinolaryngology, gynecology, and gastroenterology for various epithelia and cancerous tissues (D'Hallewin et al., 1998; Guyon et al., 2012; Loning et al., 2004; Piotrowski et al., 2004). In the USA, it has been used with the approval of the Food and Drug Administration (FDA) as a therapeutic drug for solar keratosis. Its use has not yet been approved by the pharmaceutical authority in Japan, and it is only used after obtaining approval from the ethics committee of the respective institutes. The tumor tissue concentra‐ tion of the fluorescent dye peaks at 2–6 hours after oral administration and disappears by 12 hours. Administration of antacid should be avoided at the time of oral administration be‐ cause the dye is easily decomposed in the presence of alkali, and use of 5-ALA is contraindi‐ cated in cases of porphyria, a genetic disease. PpIX biosynthesized from 5-ALA is a phototoxic substance, although the incidence of skin photosensitivity is lower than that re‐

cal issues, especially photokilling of residual neoplastic cells.

250 Clinical Management and Evolving Novel Therapeutic Strategies for Patients with Brain Tumors

triggered photodamage in glioma patients (Figure 1).

**2. General knowledge of 5–ALA**

5-ALA, the metabolic precursor of heme in the heme biosynthesis pathway, is not itself fluorescent, but is metabolized into endogenous fluorescent PpIX (Gossner et al., 1998; Inoue et al., 2007; Kennedy & Pottier, 1992; Loh et al., 1993; Peng et al., 1997). Heme biosynthesis consists of a series of enzyme-catalyzed steps, involving 5-aminolevulinate synthases 1 and 2, 5-aminolevulinate dehydratase, hydroxymethylbilane synthase, uroporphyrinogen III synthase, uroporphyrinogen decarboxylase, coproporphyrinogen oxidase (CPOX), protopor‐ phyrinogen oxidase, and ferrochelatase (FECH). In addition to these enzymes, several transporters are also involved in the biosynthesis and catabolism of heme and porphyrin, including oligopeptide transporters 1 and 2 (PEPT1 and PEPT2) and the ABC transporters ABCB6 and ABCG2 (Takahashi et al., 2011) (Figure 2).

Subsequently, biliverdin is metabolized to bilirubin by biliverdin reductase. The PEPT1/PEPT2 (influx transporters) can transport exogenous ALA from the extracellular space into the cytoplasm of target cells. ABCB6 is the ABC transporter responsible for the import of copro‐ porphyrinogen III into the mitochondria, whereas ABCG2 transports PpIX across the plasma

Current Applications of 5–ALA in Glioma Diagnostics and Therapy

http://dx.doi.org/10.5772/52428

253

Oral-intake of 5-ALA results in the accumulation of PpIX in malignant gliomas. Because of the presence of the blood-brain barrier (BBB), 5-ALA given via the oral route does not usually enter the normal brain tissue. However, it can easily pass through the disrupted BBB found in the glioma tissue (Stummer et al., 2003). PpIX that accumulates in the intracellular compartments of tumor cells exhibits red fluorescence under excitation light of an appropriate wavelength (Takahashi et al., 2011). PpIX-accumulating tumor cells can be visually discerned intraopera‐ tively from the surrounding normal cells that accumulate PpIX to a much lesser extent. To date, little is known about the molecular mechanisms underlying PpIX accumulation in malignant brain tumors after administration of 5-ALA; despite this, in the past decade, studies on ALA focusing on the mechanism of 5-ALA uptake in glioma tissue have been conducted by many research groups. Previously, our group revealed significantly greater down-regula‐ tion of FECH expression in glioblastomas than in normal brain tissues and that FECH plays a role in the metabolism of 5-ALA in glioma (Teng et al., 2011). In addition, Takahashi et al. demonstrated that the upregulated expression of the CPOX gene is correlated with the intensity of tumor fluorescence induced by 5-ALA; they also found that the mRNA level of ABCG2 was somewhat lower in the brain tumors with high ALA-induced fluorescence than in those without ALA-induced fluorescence (Takahashi et al., 2011). Therefore, they assumed that both the induction of the CPOX gene and the inhibition of ABCG2 would increase 5-ALAinduced PpIX accumulation and thereby enhance the efficacy of 5-ALA-PDT in malignant brain tumors. In agreement with this hypothesis, Zhao et al. demonstrated that ABCB6 expression levels were greatly elevated in human gliomas than in normal brain tissues and correlated with glioma histologic grade, indicating a crucial role for ABCB6 in ALA metabo‐

Further studies are required to explore the detailed mechanism of 5-ALA uptake and improve

Many previous reports describing prognostic factors in patients with malignant gliomas indicate that outcome is associated with the completeness of tumor removal (Stummer et al., 1998a). Improving the prognosis of glioma patients cannot be achieved without considerable effort to remove as much as of the lesion as possible. Since glioma tissue is not easily recognized intraoperatively, methods that easily detect tumor margins during surgery would be extreme‐ ly beneficial. 5-ALA fluorescence-guided resection is a rapidly growing, novel approach in treating glioma patients to improve the extent of tumor resection with broad applications in both preclinical and clinical settings (Stummer et al., 2000; Stummer et al., 1998b; Stummer et

membrane to maintain intracellular PpIX homeostasis (Takahashi et al., 2011).

lism and accumulation of PpIX in gliomas (Zhao et al., 2012).

our knowledge concerning 5-ALA biology in gliomas.

**4. 5–ALA guided neurosurgery**

**Figure 2.** The porphyrin-heme biosynthetic pathway and putative mitochondrial transporters. The first step of heme synthesis occurs in the mitochondrial matrix with the condensation of succinyl CoA and glycine by ALA synthase to generate ALA. Oligopeptide transporters (PEPT1 or PEPT2) are responsible for the import of exogenous ALA from the extracellular space into the cytoplasm of target cells. ALA is converted to coproporphyrinogen III by 4 enzymatic reac‐ tions. Then, coproporphyrinogen III is transported back into the mitochondrial intermembrane space (IMS), possibly via ABCB6, where it is converted to protoporphyrinogen by protoporphyrinogen III oxidase. The conversion of proto‐ porphyrinogen to PpIX by protoporphyrinogen oxidase, its transport into the matrix and the addition of Fe2+ by ferro‐ chelatase (FECH) to generate heme are coupled processes. The transporter responsible for heme transfer across the outer mitochondrial membrane remains unidentified. Heme formed from porphyrin is catabolized to biliverdin by the microsomal enzyme heme oxygenase 1. Biliverdin is subsequently metabolized to bilirubin by biliverdin reductase. ABCG2 transports porphyrins across the plasma membrane to maintain intracellular porphyrin homeostasis.

Porphyrins are synthesized from glycine and succinyl CoA via a series of enzymatic reactions. The last enzymatic reaction is the conversion of PpIX to heme by FECH, which is located in the inner mitochondrial membrane (Ferreira et al., 1995; Kemmner et al., 2008). Heme formed from porphyrin is catabolized to biliverdin by the microsomal enzyme heme oxygenase 1. Subsequently, biliverdin is metabolized to bilirubin by biliverdin reductase. The PEPT1/PEPT2 (influx transporters) can transport exogenous ALA from the extracellular space into the cytoplasm of target cells. ABCB6 is the ABC transporter responsible for the import of copro‐ porphyrinogen III into the mitochondria, whereas ABCG2 transports PpIX across the plasma membrane to maintain intracellular PpIX homeostasis (Takahashi et al., 2011).

Oral-intake of 5-ALA results in the accumulation of PpIX in malignant gliomas. Because of the presence of the blood-brain barrier (BBB), 5-ALA given via the oral route does not usually enter the normal brain tissue. However, it can easily pass through the disrupted BBB found in the glioma tissue (Stummer et al., 2003). PpIX that accumulates in the intracellular compartments of tumor cells exhibits red fluorescence under excitation light of an appropriate wavelength (Takahashi et al., 2011). PpIX-accumulating tumor cells can be visually discerned intraopera‐ tively from the surrounding normal cells that accumulate PpIX to a much lesser extent. To date, little is known about the molecular mechanisms underlying PpIX accumulation in malignant brain tumors after administration of 5-ALA; despite this, in the past decade, studies on ALA focusing on the mechanism of 5-ALA uptake in glioma tissue have been conducted by many research groups. Previously, our group revealed significantly greater down-regula‐ tion of FECH expression in glioblastomas than in normal brain tissues and that FECH plays a role in the metabolism of 5-ALA in glioma (Teng et al., 2011). In addition, Takahashi et al. demonstrated that the upregulated expression of the CPOX gene is correlated with the intensity of tumor fluorescence induced by 5-ALA; they also found that the mRNA level of ABCG2 was somewhat lower in the brain tumors with high ALA-induced fluorescence than in those without ALA-induced fluorescence (Takahashi et al., 2011). Therefore, they assumed that both the induction of the CPOX gene and the inhibition of ABCG2 would increase 5-ALAinduced PpIX accumulation and thereby enhance the efficacy of 5-ALA-PDT in malignant brain tumors. In agreement with this hypothesis, Zhao et al. demonstrated that ABCB6 expression levels were greatly elevated in human gliomas than in normal brain tissues and correlated with glioma histologic grade, indicating a crucial role for ABCB6 in ALA metabo‐ lism and accumulation of PpIX in gliomas (Zhao et al., 2012).

Further studies are required to explore the detailed mechanism of 5-ALA uptake and improve our knowledge concerning 5-ALA biology in gliomas.

### **4. 5–ALA guided neurosurgery**

5-aminolevulinate dehydratase, hydroxymethylbilane synthase, uroporphyrinogen III synthase, uroporphyrinogen decarboxylase, coproporphyrinogen oxidase (CPOX), protopor‐ phyrinogen oxidase, and ferrochelatase (FECH). In addition to these enzymes, several transporters are also involved in the biosynthesis and catabolism of heme and porphyrin, including oligopeptide transporters 1 and 2 (PEPT1 and PEPT2) and the ABC transporters

ABCB6

**Figure 2.** The porphyrin-heme biosynthetic pathway and putative mitochondrial transporters. The first step of heme synthesis occurs in the mitochondrial matrix with the condensation of succinyl CoA and glycine by ALA synthase to generate ALA. Oligopeptide transporters (PEPT1 or PEPT2) are responsible for the import of exogenous ALA from the extracellular space into the cytoplasm of target cells. ALA is converted to coproporphyrinogen III by 4 enzymatic reac‐ tions. Then, coproporphyrinogen III is transported back into the mitochondrial intermembrane space (IMS), possibly via ABCB6, where it is converted to protoporphyrinogen by protoporphyrinogen III oxidase. The conversion of proto‐ porphyrinogen to PpIX by protoporphyrinogen oxidase, its transport into the matrix and the addition of Fe2+ by ferro‐ chelatase (FECH) to generate heme are coupled processes. The transporter responsible for heme transfer across the outer mitochondrial membrane remains unidentified. Heme formed from porphyrin is catabolized to biliverdin by the microsomal enzyme heme oxygenase 1. Biliverdin is subsequently metabolized to bilirubin by biliverdin reductase.

ABCG2 transports porphyrins across the plasma membrane to maintain intracellular porphyrin homeostasis.

Porphyrins are synthesized from glycine and succinyl CoA via a series of enzymatic reactions. The last enzymatic reaction is the conversion of PpIX to heme by FECH, which is located in the inner mitochondrial membrane (Ferreira et al., 1995; Kemmner et al., 2008). Heme formed from porphyrin is catabolized to biliverdin by the microsomal enzyme heme oxygenase 1.

ALA dehydratase

ALA

PEPT1 or 2

ABCG2

Delta-ALA

Porphobilinogen (PBG)

PBG deaminase

Hydroxymethylbilane

Uroporphyrinogen III synthase

Uroporphyrinogen III

Uroporphyrinogen decarboxylase

Coproporphyrinogen III

Heme

Heme oxygenase I

Protoporphyrin IX

Biliverdin

Biliverdin reductase

Bilirubin

Cytoplasm

ABCB6 and ABCG2 (Takahashi et al., 2011) (Figure 2).

252 Clinical Management and Evolving Novel Therapeutic Strategies for Patients with Brain Tumors

ALA synthase

Coproporphyrinogen III

Succinyl CoA ALA

Glycine

+Fe2+

Coproporphyrinogen III oxidase

Protoporphyrinogen

Protoporphyrinogen oxidase

Protoporphyrin IX

Ferrochelatase

Heme

Mitochondrion

Many previous reports describing prognostic factors in patients with malignant gliomas indicate that outcome is associated with the completeness of tumor removal (Stummer et al., 1998a). Improving the prognosis of glioma patients cannot be achieved without considerable effort to remove as much as of the lesion as possible. Since glioma tissue is not easily recognized intraoperatively, methods that easily detect tumor margins during surgery would be extreme‐ ly beneficial. 5-ALA fluorescence-guided resection is a rapidly growing, novel approach in treating glioma patients to improve the extent of tumor resection with broad applications in both preclinical and clinical settings (Stummer et al., 2000; Stummer et al., 1998b; Stummer et

al., 1998c). In 1998, Stummer et al. first presented a detailed description of the technical principles for 5-ALA fluorescence-guided microsurgical resection of malignant glioma tissue (Stummer et al., 1998a). Subsequently, they performed a prospective study in 52 glioblastoma patients and identified the usefulness of 5-ALA-induced tumor fluorescence for guiding tumor resection. Recent controlled Phase III clinical trials have demonstrated that compared to conventional microsurgery, 5-ALA fluorescence-guided microsurgical resection enables more complete resection of contrast-enhancing lesions and improves progression-free survival in patients with malignant glioma (Pichlmeier et al., 2008; Stummer et al., 2006). After the rapid development of the neurosurgical fluorescence operation microscope, several investigators have recently demonstrated the feasibility of 5-ALA fluorescence-guided resection in patients with glioma (Kuroiwa et al., 1998; Kuroiwa et al., 2001; Sherman et al., 2011). Widhalm et al. also indicated that 5-ALA is a promising marker for intraoperative visualization of anaplastic foci in diffusely infiltrating gliomas without contrast enhancement and showed that the positive predictive value (PVV) of focal 5-ALA fluorescence for World Health Organization (WHO) grade III glioma was 100% (sensitivity 89%) (Widhalm et al., 2010). In addition, Nabavi's group investigated the feasibility and selectivity of 5-ALA-induced fluorescence to guide resection in recurrent gliomas (WHO grade III/IV) and found that 5-ALA fluorescence (weak and strong) had a high PPV (97.2%) in all pathological-appearing tissues obtained from 354 biopsies performed in 36 patients (Nabavi et al., 2009). Based on these findings, they suggested that 5-ALA fluorescence guidance is an effective surgical adjunct in the surgery of recurrent malignant gliomas, and many other trials have displayed similar successful results using 5-ALA fluorescence-guided resection in meningiomas to achieve optimal resection (Coluccia et al., 2010; Kajimoto et al., 2007).

5-ALA has become widely accepted. 5-ALA-PDT is a novel treatment modality for early or superficial cancers, as well as a palliative treatment to a certain extent, that triggers a photo‐ dynamic effect similar to that of light in the visible range to produce a rapid PDT response in the targeted tissue. Three types of mechanisms have been identified in the literature as contributing to the rapid PDT response *in vivo*: (1) PDT can directly damage and kill the malignant tumor cells, either by apoptosis or a non-apoptotic mechanism; (2) PDT may produce profound changes in the tumor vasculature, including blood flow stasis, vascular collapse, and/or vascular leakage, that can result in indirect killing of malignant cells; and (3) PDT can promote the release of cytokines and other inflammatory mediators from target cells that induce an inflammatory response and recruit additional host cells to the tumor (Mac‐ Donald & Dougherty, 2001; Oleinick et al., 2002). Recent studies have shown that 5-ALA-PDTinduced photodamage causes mitochondrial and nuclear DNA damage. As a result, massive apoptosis occurs owing to mitochondrial release of cytochrome c and activation of caspase-3 and caspase-9 in glioma cells (Inoue et al., 2007; Karmakar et al., 2007). These results indicate

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Compared to the other modalities, 5-ALA-PDT has the advantage of better specificity and lower complication rates, and it is also a good alternative in tumor patients not eligible for surgery. Unfortunately, current 5-ALA-PDT protocols have yet to be widely established in clinical treatment for glioma. This may be partly due to limitations in current PDT regimens and partly due to the therapeutic efficacy of 5-ALA-PDT in preclinical settings (Teng et al., 2011). The reason for 5-ALA-PDT not being standard treatment in malignant glioma may be multifactorial, including the lack of randomized controlled trials and an optimal 5-ALA-PDT regimen. Generally, PDT efficacy depends on parameters such as the photosensitizing agent, irradiance and timing, oxygen, photosensitizer concentration, and different pathologic grade glioma tissue sensitivity to the PDT effect (Teng et al., 2011). Optimizing these parameters is difficult, expensive, and time consuming. The efficacy of 5-ALA-PDT may also be limited by reduced penetration of appropriate light through the target tissue and local acute phototoxicity to normal surrounding brain tissue (Norum et al., 2009). In addition, post-treatment edema and long-lasting skin photosensitivity after PDT of brain tumors are potentially challenging side effects that neurosurgeons have to deal with. Regarding the specificity of PDD for glioma surgery, new techniques allowing more specific accumulation of PpIX in target tumor cells need to be developed. Further studies are required for screening available and specific glioma antigens that are strongly expressed in glioma tissues, but not in normal brain tissues. 5-ALA labeled with antibodies against glioma-specific antigens may be useful in increasing the accumulation of PpIX and the specificity of PDD in glioma tissues. If the specificity and selectivity of 5-ALA-PDT could be improved in glioma tissue, 5-ALA-PDT would certainly be

that 5-ALA-PDT triggers apoptosis through a mitochondrial pathway.

regarded as a promising and competitive alternative in glioma treatment.

In this review, we focused on the 5-ALA agent as a potential treatment modality for patients with malignant glioma. 5-ALA-induced fluorescence is a useful intraoperative tool for the

**6. Perspective**

Despite the advantages of 5-ALA fluorescence, successful glioma resection often depends on the neurosurgeon's ability to distinguish residual tumor tissue from surrounding brain tissue even with the assistance of PpIX fluorescence (Teng et al., 2011). The marginal area containing infiltrating glioma cells shows vague fluorescence because the density of the glioma cells in these areas is low and heterogeneous, resulting in insufficient 5-ALA uptake and PpIX accumulation (Utsuki et al., 2006). Conversely, because of the presence of reactive astrocytes and macrophages and leakage of PpIX into the extracellular matrix, false-positive (i.e., the area fluoresced, but no tumor was seen histologically) results in weakly fluorescing normalappearing tissue are also common (Utsuki et al., 2007). Therefore, more objective and quanti‐ tative indicators need to be established for neurosurgeons to improve intraoperative identification of tumor margins. Recently, a quantitative method involving spectroscopic analysis for determining the amount of fluorescence present has been developed, which allows more precise visualization of quantitative fluorescence from tumor tissues (Utsuki et al., 2006; Valdes et al., 2011).

### **5. 5–ALA mediated-PDT**

PDT is a tool for the treatment of certain cancerous and pre-cancerous conditions, especially in the field of dermatology (Buytaert et al., 2007). Among the many kinds of photosensitizers, 5-ALA has become widely accepted. 5-ALA-PDT is a novel treatment modality for early or superficial cancers, as well as a palliative treatment to a certain extent, that triggers a photo‐ dynamic effect similar to that of light in the visible range to produce a rapid PDT response in the targeted tissue. Three types of mechanisms have been identified in the literature as contributing to the rapid PDT response *in vivo*: (1) PDT can directly damage and kill the malignant tumor cells, either by apoptosis or a non-apoptotic mechanism; (2) PDT may produce profound changes in the tumor vasculature, including blood flow stasis, vascular collapse, and/or vascular leakage, that can result in indirect killing of malignant cells; and (3) PDT can promote the release of cytokines and other inflammatory mediators from target cells that induce an inflammatory response and recruit additional host cells to the tumor (Mac‐ Donald & Dougherty, 2001; Oleinick et al., 2002). Recent studies have shown that 5-ALA-PDTinduced photodamage causes mitochondrial and nuclear DNA damage. As a result, massive apoptosis occurs owing to mitochondrial release of cytochrome c and activation of caspase-3 and caspase-9 in glioma cells (Inoue et al., 2007; Karmakar et al., 2007). These results indicate that 5-ALA-PDT triggers apoptosis through a mitochondrial pathway.

Compared to the other modalities, 5-ALA-PDT has the advantage of better specificity and lower complication rates, and it is also a good alternative in tumor patients not eligible for surgery. Unfortunately, current 5-ALA-PDT protocols have yet to be widely established in clinical treatment for glioma. This may be partly due to limitations in current PDT regimens and partly due to the therapeutic efficacy of 5-ALA-PDT in preclinical settings (Teng et al., 2011). The reason for 5-ALA-PDT not being standard treatment in malignant glioma may be multifactorial, including the lack of randomized controlled trials and an optimal 5-ALA-PDT regimen. Generally, PDT efficacy depends on parameters such as the photosensitizing agent, irradiance and timing, oxygen, photosensitizer concentration, and different pathologic grade glioma tissue sensitivity to the PDT effect (Teng et al., 2011). Optimizing these parameters is difficult, expensive, and time consuming. The efficacy of 5-ALA-PDT may also be limited by reduced penetration of appropriate light through the target tissue and local acute phototoxicity to normal surrounding brain tissue (Norum et al., 2009). In addition, post-treatment edema and long-lasting skin photosensitivity after PDT of brain tumors are potentially challenging side effects that neurosurgeons have to deal with. Regarding the specificity of PDD for glioma surgery, new techniques allowing more specific accumulation of PpIX in target tumor cells need to be developed. Further studies are required for screening available and specific glioma antigens that are strongly expressed in glioma tissues, but not in normal brain tissues. 5-ALA labeled with antibodies against glioma-specific antigens may be useful in increasing the accumulation of PpIX and the specificity of PDD in glioma tissues. If the specificity and selectivity of 5-ALA-PDT could be improved in glioma tissue, 5-ALA-PDT would certainly be regarded as a promising and competitive alternative in glioma treatment.

### **6. Perspective**

al., 1998c). In 1998, Stummer et al. first presented a detailed description of the technical principles for 5-ALA fluorescence-guided microsurgical resection of malignant glioma tissue (Stummer et al., 1998a). Subsequently, they performed a prospective study in 52 glioblastoma patients and identified the usefulness of 5-ALA-induced tumor fluorescence for guiding tumor resection. Recent controlled Phase III clinical trials have demonstrated that compared to conventional microsurgery, 5-ALA fluorescence-guided microsurgical resection enables more complete resection of contrast-enhancing lesions and improves progression-free survival in patients with malignant glioma (Pichlmeier et al., 2008; Stummer et al., 2006). After the rapid development of the neurosurgical fluorescence operation microscope, several investigators have recently demonstrated the feasibility of 5-ALA fluorescence-guided resection in patients with glioma (Kuroiwa et al., 1998; Kuroiwa et al., 2001; Sherman et al., 2011). Widhalm et al. also indicated that 5-ALA is a promising marker for intraoperative visualization of anaplastic foci in diffusely infiltrating gliomas without contrast enhancement and showed that the positive predictive value (PVV) of focal 5-ALA fluorescence for World Health Organization (WHO) grade III glioma was 100% (sensitivity 89%) (Widhalm et al., 2010). In addition, Nabavi's group investigated the feasibility and selectivity of 5-ALA-induced fluorescence to guide resection in recurrent gliomas (WHO grade III/IV) and found that 5-ALA fluorescence (weak and strong) had a high PPV (97.2%) in all pathological-appearing tissues obtained from 354 biopsies performed in 36 patients (Nabavi et al., 2009). Based on these findings, they suggested that 5-ALA fluorescence guidance is an effective surgical adjunct in the surgery of recurrent malignant gliomas, and many other trials have displayed similar successful results using 5-ALA fluorescence-guided resection in meningiomas to achieve optimal resection

254 Clinical Management and Evolving Novel Therapeutic Strategies for Patients with Brain Tumors

Despite the advantages of 5-ALA fluorescence, successful glioma resection often depends on the neurosurgeon's ability to distinguish residual tumor tissue from surrounding brain tissue even with the assistance of PpIX fluorescence (Teng et al., 2011). The marginal area containing infiltrating glioma cells shows vague fluorescence because the density of the glioma cells in these areas is low and heterogeneous, resulting in insufficient 5-ALA uptake and PpIX accumulation (Utsuki et al., 2006). Conversely, because of the presence of reactive astrocytes and macrophages and leakage of PpIX into the extracellular matrix, false-positive (i.e., the area fluoresced, but no tumor was seen histologically) results in weakly fluorescing normalappearing tissue are also common (Utsuki et al., 2007). Therefore, more objective and quanti‐ tative indicators need to be established for neurosurgeons to improve intraoperative identification of tumor margins. Recently, a quantitative method involving spectroscopic analysis for determining the amount of fluorescence present has been developed, which allows more precise visualization of quantitative fluorescence from tumor tissues (Utsuki et al.,

PDT is a tool for the treatment of certain cancerous and pre-cancerous conditions, especially in the field of dermatology (Buytaert et al., 2007). Among the many kinds of photosensitizers,

(Coluccia et al., 2010; Kajimoto et al., 2007).

2006; Valdes et al., 2011).

**5. 5–ALA mediated-PDT**

In this review, we focused on the 5-ALA agent as a potential treatment modality for patients with malignant glioma. 5-ALA-induced fluorescence is a useful intraoperative tool for the visualization of glioma tissue, and 5-ALA-PDT is a promising and alternative adjuvant therapy for photokilling residual neoplastic cells. Although impressive advances in the application of 5-ALA to glioma have been made from many clinical trials, this evolving field still faces important challenges. For example, single imaging in solid malignant glioma tissue with all of these features is not sufficient. Judgment of the extent of resection may still be treacherous within infiltrating parts of brain tumors, where the ratio of background noise of normal brain tissue to signal intensity of PpIX-saturated malignant glioma cells is less pronounced (Hefti et al., 2012; Liao et al., 2012). Recent efforts to boost target-to-background ratios have used a combination of intraoperative 5-ALA-induced fluorescence and 3D MRI imaging or neurona‐ vigation imaging with some success (Liao et al., 2008; Panciani et al., 2012). Furthermore, more objective indicators to measure quantitative PpIX concentrations intraoperatively in the brain tumor margin need to be established. In the future, laboratory and clinical studies should be devoted to 5-ALA-PDT in conjunction with the use of other therapies, which will have maximal effect on the residual tumor after resection. This multiple adjuvant therapy should enhance specificity and allow lowering of the total PDT dose, while still increasing the therapeutic efficacy of PDT. Other proposals should adjust the current PDT treatment regimens, modify the existing photosensitizer, or develop new and more specific photosensitizers. Further studies are needed to elucidate possible mechanisms of 5-ALA uptake that could explain the diversity of intraoperative findings.

[2] Babu, R, & Adamson, C. (2012). Fluorescence-Guided Malignant Glioma Resections.

Current Applications of 5–ALA in Glioma Diagnostics and Therapy

http://dx.doi.org/10.5772/52428

257

[3] Blake, E, & Curnow, A. (2010). The hydroxypyridinone iron chelator CP94 can en‐ hance PpIX-induced PDT of cultured human glioma cells. Photochem Photobiol.

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### **Author details**

Lei Teng1,2, Mitsutoshi Nakada1\*, Yutaka Hayashi1 , Takeshi Yoneyama3 , Shi-Guang Zhao2 and Jun-Ichiro Hamada1

\*Address all correspondence to<EMAIL_ADDRESS>
1 Department of Neurosurgery, Graduate School of Medical Science, Kanazawa University, Kanazawa,, Japan

2 Department of Neurosurgery, The First Clinical College of Harbin Medical University, Harbin,, China

3 College of Science and Engineering, Kanazawa University, Kanazawa,, Japan

### **References**

[1] Aydin, H, Sillenberg, I, & Von Lieven, H. (2001). Patterns of failure following CTbased 3-D irradiation for malignant glioma. Strahlenther Onkol. 0179-7158, 177(8), 424-431.

[2] Babu, R, & Adamson, C. (2012). Fluorescence-Guided Malignant Glioma Resections. Curr Drug Discov Technol. Vol. No. pp. 1875-6220, 1875-6220.

visualization of glioma tissue, and 5-ALA-PDT is a promising and alternative adjuvant therapy for photokilling residual neoplastic cells. Although impressive advances in the application of 5-ALA to glioma have been made from many clinical trials, this evolving field still faces important challenges. For example, single imaging in solid malignant glioma tissue with all of these features is not sufficient. Judgment of the extent of resection may still be treacherous within infiltrating parts of brain tumors, where the ratio of background noise of normal brain tissue to signal intensity of PpIX-saturated malignant glioma cells is less pronounced (Hefti et al., 2012; Liao et al., 2012). Recent efforts to boost target-to-background ratios have used a combination of intraoperative 5-ALA-induced fluorescence and 3D MRI imaging or neurona‐ vigation imaging with some success (Liao et al., 2008; Panciani et al., 2012). Furthermore, more objective indicators to measure quantitative PpIX concentrations intraoperatively in the brain tumor margin need to be established. In the future, laboratory and clinical studies should be devoted to 5-ALA-PDT in conjunction with the use of other therapies, which will have maximal effect on the residual tumor after resection. This multiple adjuvant therapy should enhance specificity and allow lowering of the total PDT dose, while still increasing the therapeutic efficacy of PDT. Other proposals should adjust the current PDT treatment regimens, modify the existing photosensitizer, or develop new and more specific photosensitizers. Further studies are needed to elucidate possible mechanisms of 5-ALA uptake that could explain the

256 Clinical Management and Evolving Novel Therapeutic Strategies for Patients with Brain Tumors

, Takeshi Yoneyama3

, Shi-Guang Zhao2

and

diversity of intraoperative findings.

Lei Teng1,2, Mitsutoshi Nakada1\*, Yutaka Hayashi1

\*Address all correspondence to<EMAIL_ADDRESS>
1 Department of Neurosurgery, Graduate School of Medical Science, Kanazawa University,

2 Department of Neurosurgery, The First Clinical College of Harbin Medical University,

[1] Aydin, H, Sillenberg, I, & Von Lieven, H. (2001). Patterns of failure following CTbased 3-D irradiation for malignant glioma. Strahlenther Onkol. 0179-7158, 177(8),

3 College of Science and Engineering, Kanazawa University, Kanazawa,, Japan

**Author details**

Jun-Ichiro Hamada1

Kanazawa,, Japan

Harbin,, China

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0065-1419, 88, 9-12.

1011-1344, 45(2-3), 160-169.

104(5), 798-807.

0470-8105, 47(5), 210-213.

surg. 0022-3085, 104(4), 618-620.

525-526, 0014-8396X, 42(3), 518-525.

**Chapter 13**

**Photodynamic Therapy Using Talaporfin Sodium and**

**Diode Laser for Newly Diagnosed Malignant Gliomas**

Wilson et al. [1] investigated the progression of glioblastoma (Grade 4) at the cellular level and showed that cells from tumors of 4 cm or greater in diameter on diagnostic imaging are considered to be present in both normal and affected brain tissues, with a distribution of 92% of tumor cells in the tumor bulk, 6% in areas within a 2-cm margin around the tumor, and 1.8% within an additional 2-cm area surrounding this margin. The majority of recurrent malignant gliomas develop in this marginal area, and therefore, controlling infiltrating cells in this region while protecting normal brain cells is critical to inhibit tumor recurrence.

In photodynamic therapy (PDT), a photosensitizer taken up in tumor tissues and neovascular‐ ized tumor vessels is administered to cancer patients. Tumor tissues are then later irradiated using a laser to induce a photochemical reaction in the photosensitizer, thereby selectively causing tumor cell apoptosis and necrosis. This tumor cell apoptosis and necrosis result from the strong oxidative effect of singlet oxygen (active oxygen) produced by the photochemical re‐

We previously reported that PDT using talaporfin sodium (TS), a second-generation photo‐ sensitizer, induced tumor coagulation necrosis and tumor cell-selective apoptosis in rats in which C6 glioma cells had been transplanted into the brain [3]. In the present study, we evaluated the safety and efficacy of PDT in areas with tumor invasion following tumor re‐

Eligible subjects were patients with a first time diagnosis of adult intracranial malignant glioma (Grade 3 or 4) based on preoperative diagnostic imaging and with a tumor bulk adja‐

and reproduction in any medium, provided the original work is properly cited.

© 2013 Akimoto; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use,

© 2013 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution,

distribution, and reproduction in any medium, provided the original work is properly cited.

action in the photosensitizer, induced by a laser at a specific wavelength [2].

moval in patients with malignant glioma.

**2. Materials**

Jiro Akimoto

**1. Introduction**

http://dx.doi.org/10.5772/53485

Additional information is available at the end of the chapter

## **Photodynamic Therapy Using Talaporfin Sodium and Diode Laser for Newly Diagnosed Malignant Gliomas**

Jiro Akimoto

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/53485

### **1. Introduction**

Wilson et al. [1] investigated the progression of glioblastoma (Grade 4) at the cellular level and showed that cells from tumors of 4 cm or greater in diameter on diagnostic imaging are considered to be present in both normal and affected brain tissues, with a distribution of 92% of tumor cells in the tumor bulk, 6% in areas within a 2-cm margin around the tumor, and 1.8% within an additional 2-cm area surrounding this margin. The majority of recurrent malignant gliomas develop in this marginal area, and therefore, controlling infiltrating cells in this region while protecting normal brain cells is critical to inhibit tumor recurrence.

In photodynamic therapy (PDT), a photosensitizer taken up in tumor tissues and neovascular‐ ized tumor vessels is administered to cancer patients. Tumor tissues are then later irradiated using a laser to induce a photochemical reaction in the photosensitizer, thereby selectively causing tumor cell apoptosis and necrosis. This tumor cell apoptosis and necrosis result from the strong oxidative effect of singlet oxygen (active oxygen) produced by the photochemical re‐ action in the photosensitizer, induced by a laser at a specific wavelength [2].

We previously reported that PDT using talaporfin sodium (TS), a second-generation photo‐ sensitizer, induced tumor coagulation necrosis and tumor cell-selective apoptosis in rats in which C6 glioma cells had been transplanted into the brain [3]. In the present study, we evaluated the safety and efficacy of PDT in areas with tumor invasion following tumor re‐ moval in patients with malignant glioma.

### **2. Materials**

Eligible subjects were patients with a first time diagnosis of adult intracranial malignant glioma (Grade 3 or 4) based on preoperative diagnostic imaging and with a tumor bulk adja‐

cent to the eloquent areas of the brain associated with language, motor, sensation and vi‐ sion, for which a wide resection of the tumor much larger than the tumor bulk was impossible. Selective treatment of the infiltrating tumor cells by PDT was considered neces‐ sary because of tumor invasion into the eloquent areas, despite the fact that the tumor bulk had been removed as extensively as possible. PDT was performed after a rapid intraopera‐ tive pathological diagnosis confirmed malignant glioma. We excluded patients who had re‐ ceived radiotherapy or chemotherapy within 3 weeks prior to the surgery, patients with bleeding or abnormal coagulation test results, and patients with porphyria or porphyrin hy‐ persensitivity. This clinical study was approved by the Institutional Review Board of our in‐ stitution. All subjects provided written informed consent. The subjects were consecutive 8 patients (7 men and 1 woman) treated at our hospital between May 2006 and Mar 2009 and aged 48 to 82 years (median, 58 years). The tumors were localized in the frontal lobe in 4 patients, temporal lobe in 2 patients, insular gyrus in 1 patient and parietal lobe in 1 patient. Preoperative Karnofsky Performance Scale (KPS) was 50 to 80 (median 60). The extent of tu‐ mor resection was subtotal in 7 patients and partial in 1 patient. The final pathological diag‐ nosis was Grade 3 in 3 patients, and Grade 4 glioblastoma in 5 patients. As postoperative adjuvant therapy, 4 patients received irradiation in combination with temozolomide (TMZ) and 2 patients received irradiation and ACNU-based multidrug chemotherapy.

### **3. Methods**

#### **3.1. Talaporfin sodium and diode laser**

Talaporfin sodium (TS: Laserphyrin®, Meiji Seika Pharma Co., Ltd., Tokyo, Japan) is a photosensitizer utilized in PDT which was approved for use in Japan in 2003 as treat‐ ment for early-stage lung cancer, in conjunction with a diode laser (PD Laser®, Panasonic HealthCare Co., Ltd., Ehime, Japan) at a wavelength of 664 nm [4]. (Fig. 1A and B) Tala‐ porfin sodium is a second-generation photosensitizer which is more quickly excreted from the body than the first-generation porfimer sodium (Photofrin®, Pfizer Japan Inc.,Tokyo, Japan). It is characterized by the rapid resolution of a skin photosensitive reaction, which is important with the use of photosensitizers. This has enabled the period of necessary light shielding in a room with measurement of < 500 lux to be reduced to 2 weeks for ta‐ laporfin sodium, whereas porfimer sodium requires patients to stay in a semi-dark(< 300 lux) room for 1 month after administration. The diode laser instrument is a compact sys‐ tem weighing 14 kg, has low power consumption, and can be easily maintained without the need for dye exchange. (Fig. 2A).

(a)

Photodynamic Therapy Using Talaporfin Sodium and Diode Laser for Newly Diagnosed Malignant Gliomas

http://dx.doi.org/10.5772/53485

265

(b)

**Figure 1.** Second-generation photosensitizer, Talaporfin sodium. A:Chemical structures of talaporfin sodium (mono-Laspartyl chlorine e6, NPe6) *N*-[[(2S,3S)-18-carboxy-2-(2-carboxyethyl)-13-ethyl-2,3-dibydro-3,7,12,17-tetramethyl-8-vi‐ nylporphyrin-20-yl]-L-asparatic acid. B:Absorption spectrum of talaporfin sodium and change in absorption wavelength following conjugation with albumin (solid line; talaporfin sodium and phosphate buffer solution, dotted line; talaporfin sodium conjugated with albumin) talaporfin sodium has absorption peaks in the Soret band (398nm) and Q bands (502, 530, 620 and 654nm) in pH 7.4 phosphate buffer solution (PBS). When it conjugates with albumin,

its absorption band wavelength becomes approximately 10nm longer (bathochromic shift).

#### **3.2. Operating microscope equipped with diode laser**

The PD Laser® (Panasonic HealthCare Co., Ltd.), installed in the MM30 microscope (Mitaka Kohki Co., Ltd., Tokyo, Japan), is combined into a system which provides laser irradiation from the operating microscope from a plane nearly coaxial to the surgical view. The laser is introduced into the microscope by a quartz fiber, and providing a laser transmission path close to an observation light path using a conventional halogen light. This allows surgeons to accurately identify an irradiation target area during surgery. (Fig. 2A, B)

cent to the eloquent areas of the brain associated with language, motor, sensation and vi‐ sion, for which a wide resection of the tumor much larger than the tumor bulk was impossible. Selective treatment of the infiltrating tumor cells by PDT was considered neces‐ sary because of tumor invasion into the eloquent areas, despite the fact that the tumor bulk had been removed as extensively as possible. PDT was performed after a rapid intraopera‐ tive pathological diagnosis confirmed malignant glioma. We excluded patients who had re‐ ceived radiotherapy or chemotherapy within 3 weeks prior to the surgery, patients with bleeding or abnormal coagulation test results, and patients with porphyria or porphyrin hy‐ persensitivity. This clinical study was approved by the Institutional Review Board of our in‐ stitution. All subjects provided written informed consent. The subjects were consecutive 8 patients (7 men and 1 woman) treated at our hospital between May 2006 and Mar 2009 and aged 48 to 82 years (median, 58 years). The tumors were localized in the frontal lobe in 4 patients, temporal lobe in 2 patients, insular gyrus in 1 patient and parietal lobe in 1 patient. Preoperative Karnofsky Performance Scale (KPS) was 50 to 80 (median 60). The extent of tu‐ mor resection was subtotal in 7 patients and partial in 1 patient. The final pathological diag‐ nosis was Grade 3 in 3 patients, and Grade 4 glioblastoma in 5 patients. As postoperative adjuvant therapy, 4 patients received irradiation in combination with temozolomide (TMZ)

264 Clinical Management and Evolving Novel Therapeutic Strategies for Patients with Brain Tumors

and 2 patients received irradiation and ACNU-based multidrug chemotherapy.

Talaporfin sodium (TS: Laserphyrin®, Meiji Seika Pharma Co., Ltd., Tokyo, Japan) is a photosensitizer utilized in PDT which was approved for use in Japan in 2003 as treat‐ ment for early-stage lung cancer, in conjunction with a diode laser (PD Laser®, Panasonic HealthCare Co., Ltd., Ehime, Japan) at a wavelength of 664 nm [4]. (Fig. 1A and B) Tala‐ porfin sodium is a second-generation photosensitizer which is more quickly excreted from the body than the first-generation porfimer sodium (Photofrin®, Pfizer Japan Inc.,Tokyo, Japan). It is characterized by the rapid resolution of a skin photosensitive reaction, which is important with the use of photosensitizers. This has enabled the period of necessary light shielding in a room with measurement of < 500 lux to be reduced to 2 weeks for ta‐ laporfin sodium, whereas porfimer sodium requires patients to stay in a semi-dark(< 300 lux) room for 1 month after administration. The diode laser instrument is a compact sys‐ tem weighing 14 kg, has low power consumption, and can be easily maintained without

The PD Laser® (Panasonic HealthCare Co., Ltd.), installed in the MM30 microscope (Mitaka Kohki Co., Ltd., Tokyo, Japan), is combined into a system which provides laser irradiation from the operating microscope from a plane nearly coaxial to the surgical view. The laser is introduced into the microscope by a quartz fiber, and providing a laser transmission path close to an observation light path using a conventional halogen light. This allows surgeons

to accurately identify an irradiation target area during surgery. (Fig. 2A, B)

**3. Methods**

**3.1. Talaporfin sodium and diode laser**

the need for dye exchange. (Fig. 2A).

**3.2. Operating microscope equipped with diode laser**

**Figure 1.** Second-generation photosensitizer, Talaporfin sodium. A:Chemical structures of talaporfin sodium (mono-Laspartyl chlorine e6, NPe6) *N*-[[(2S,3S)-18-carboxy-2-(2-carboxyethyl)-13-ethyl-2,3-dibydro-3,7,12,17-tetramethyl-8-vi‐ nylporphyrin-20-yl]-L-asparatic acid. B:Absorption spectrum of talaporfin sodium and change in absorption wavelength following conjugation with albumin (solid line; talaporfin sodium and phosphate buffer solution, dotted line; talaporfin sodium conjugated with albumin) talaporfin sodium has absorption peaks in the Soret band (398nm) and Q bands (502, 530, 620 and 654nm) in pH 7.4 phosphate buffer solution (PBS). When it conjugates with albumin, its absorption band wavelength becomes approximately 10nm longer (bathochromic shift).

**3.2. Operating microscope equipped with diode laser**

(Figs. 2 A, B)

The PD Laser® (Panasonic HealthCare Co., Ltd.), installed in the MM30 microscope (Mitaka Kohki Co., Ltd., Tokyo, Japan), is

light path using a conventional halogen light. This allows surgeons to accurately identify an irradiation target area during surgery.

**3.3. Photodynamic therapy (PDT)**

ing an area of 1.5 cm2

or two targets of tumor bed.

**3.4. Skin photosensitive reaction test**

exposing the retinas to direct sunlight.

(diameter of the irradiated area: 1.5 cm2

**4. Results**

J/cm2

**4.1. Selected case**

TS was administered bolus intravenously at a dose of 40mg/m2

tive microscope. Irradiation energy density of 27 J/cm2

hours prior to surgery. At surgery, maximal resection of the tumor was performed usually utilizing optical navigation system and electrophysiological monitoring leaving a tumor bed area to be irradiated. The surface area of tumor bed was observed under operative micro‐ scope and optimal navigation system, and identified the optimal target for PDT. PDT cover‐

Photodynamic Therapy Using Talaporfin Sodium and Diode Laser for Newly Diagnosed Malignant Gliomas

were to be irradiated, we took care to avoid overlapping the irradiated areas under opera‐

ation time of 180 seconds) of 664nm diode laser for PDT was irradiated superficially to one

The administration of TS enhances sensitivity to light, and which may produce a photosen‐ sitive reaction. Patients should therefore avoid direct sunlight immediately after the admin‐ istration of talaporfin sodium and stay in a room where the illumination intensity is adjusted to < 500 lux using a shading curtain. After surgery, patients were required to ex‐ pose the back of the palm of the hand to direct sunlight for 5minutes every day between 11:00 and 14:00 to evaluate the development of erythema. However, even when the result of this skin photosensitivity test confirmed the resolution of a photosensitive reaction (defined by the absence of evident erythema) and the restriction for shading at < 500 lux could be re‐ moved, patients were advised to avoid further direct sunlight for 2 weeks after the adminis‐ tration of talaporfin sodium. In particular, we recommended that special shading eye goggles should be used worn 7 days after the administration of talaporfin sodium to avoid

A 65-year-old man presented with 2 months history of headache and gradually worsened. Head MRI showed a tumor in the right frontal lobe, and edema extending as far as the premotor area at the posterior edge of the tumor bulk. On a sagittal view, part of the posterior edge of the tumor bulk extended posteriorly, and this part was determined to have invaded the supplemental motor area(SMA) (Fig. 3A and B). Using optical navigation system and motor evoked potential (MEP) monioring, we performed craniotomy in order to resect the tumor, and rapid pathological diagnosis confirmed that the tumor was a glioblastoma. From the optical navigation images, the posterior margin of the tumor bulk was confirmed to have reached the pre-motor area (SMA-proper) (Fig.3C), and a single session of PDT at 27

Under a pathological diagnosis of glioblastoma, the patient given postoperative irradiation

diameter was used for any tumor growth in these area. When 2 sites

(power density of 150mW/cm2

) was performed at this site (Fig 3.D and E).

in shielded conditions 24

http://dx.doi.org/10.5772/53485

, irradi‐

267

(a)

(b)

Figure 2. Diode laser with a neurosurgical operating microscope. A: MM80® microscope (Mitaka Kohki Co., Ltd.) equipped with a compact-size diode laser (PD Laser®, Panasonic Health Care Ltd.). B: From the operating microscope, the diode laser irradiates the surgical field with an irradiation diameter of 1.0 cm2. **Figure 2.** Diode laser with a neurosurgical operating microscope. A: MM80® microscope (Mitaka Kohki Co., Ltd.) equipped with a compact-size diode laser (PD Laser® , Panasonic Health Care Ltd., arrow). B: From the operating micro‐ scope, the diode laser irradiates the surgical field with an irradiation diameter of 1.5 cm2.

**3.3. Photodynamic therapy (PDT)**

### **3.3. Photodynamic therapy (PDT)**

**3.2. Operating microscope equipped with diode laser**

(Figs. 2 A, B)

irradiation diameter of 1.0 cm2.

**3.3. Photodynamic therapy (PDT)**

equipped with a compact-size diode laser (PD Laser®

The PD Laser® (Panasonic HealthCare Co., Ltd.), installed in the MM30 microscope (Mitaka Kohki Co., Ltd., Tokyo, Japan), is combined into a system which provides laser irradiation from the operating microscope from a plane nearly coaxial to the surgical view. The laser is introduced into the microscope by a quartz fiber, and providing a laser transmission path close to an observation light path using a conventional halogen light. This allows surgeons to accurately identify an irradiation target area during surgery.

266 Clinical Management and Evolving Novel Therapeutic Strategies for Patients with Brain Tumors

Figure 2. Diode laser with a neurosurgical operating microscope. A: MM80® microscope (Mitaka Kohki Co., Ltd.) equipped with a compact-size diode laser (PD Laser®, Panasonic Health Care Ltd.). B: From the operating microscope, the diode laser irradiates the surgical field with an

microscope (Mitaka Kohki Co., Ltd.)

, Panasonic Health Care Ltd., arrow). B: From the operating micro‐

**Figure 2.** Diode laser with a neurosurgical operating microscope. A: MM80®

scope, the diode laser irradiates the surgical field with an irradiation diameter of 1.5 cm2.

(b)

(a)

TS was administered bolus intravenously at a dose of 40mg/m2 in shielded conditions 24 hours prior to surgery. At surgery, maximal resection of the tumor was performed usually utilizing optical navigation system and electrophysiological monitoring leaving a tumor bed area to be irradiated. The surface area of tumor bed was observed under operative micro‐ scope and optimal navigation system, and identified the optimal target for PDT. PDT cover‐ ing an area of 1.5 cm2 diameter was used for any tumor growth in these area. When 2 sites were to be irradiated, we took care to avoid overlapping the irradiated areas under opera‐ tive microscope. Irradiation energy density of 27 J/cm2 (power density of 150mW/cm2 , irradi‐ ation time of 180 seconds) of 664nm diode laser for PDT was irradiated superficially to one or two targets of tumor bed.

### **3.4. Skin photosensitive reaction test**

The administration of TS enhances sensitivity to light, and which may produce a photosen‐ sitive reaction. Patients should therefore avoid direct sunlight immediately after the admin‐ istration of talaporfin sodium and stay in a room where the illumination intensity is adjusted to < 500 lux using a shading curtain. After surgery, patients were required to ex‐ pose the back of the palm of the hand to direct sunlight for 5minutes every day between 11:00 and 14:00 to evaluate the development of erythema. However, even when the result of this skin photosensitivity test confirmed the resolution of a photosensitive reaction (defined by the absence of evident erythema) and the restriction for shading at < 500 lux could be re‐ moved, patients were advised to avoid further direct sunlight for 2 weeks after the adminis‐ tration of talaporfin sodium. In particular, we recommended that special shading eye goggles should be used worn 7 days after the administration of talaporfin sodium to avoid exposing the retinas to direct sunlight.

### **4. Results**

#### **4.1. Selected case**

A 65-year-old man presented with 2 months history of headache and gradually worsened. Head MRI showed a tumor in the right frontal lobe, and edema extending as far as the premotor area at the posterior edge of the tumor bulk. On a sagittal view, part of the posterior edge of the tumor bulk extended posteriorly, and this part was determined to have invaded the supplemental motor area(SMA) (Fig. 3A and B). Using optical navigation system and motor evoked potential (MEP) monioring, we performed craniotomy in order to resect the tumor, and rapid pathological diagnosis confirmed that the tumor was a glioblastoma. From the optical navigation images, the posterior margin of the tumor bulk was confirmed to have reached the pre-motor area (SMA-proper) (Fig.3C), and a single session of PDT at 27 J/cm2 (diameter of the irradiated area: 1.5 cm2 ) was performed at this site (Fig 3.D and E). Under a pathological diagnosis of glioblastoma, the patient given postoperative irradiation and chemotherapy with oral TMZ. On day 3 after surgery, MRI showed remaining contrastenhanced tumor growth in the pre-motor area, but by the end of radiotherapy (on day 51 after surgery, Fig. 3F), complete response was confirmed at this site. His headache was dis‐ appeared and he enjoyed a play of weekly golf until 3 years after operation.

**Figure 3.** PDT and changes over time on postoperative magnetic resonance imaging (MRI) in Case 3. A: Preoperative axial FLAIR image showed the right frontal tumor and the peri-tumoral edema extending to the right pre-motor cor‐ tex. B: Preoperative gadolinium-enhanced T1-weighted sagittal imaging showed a heterogeneously enhanced tumor was present in the right superior frontal gyrus to supplementary motor cortex. C: Based on intra-operative optical nav‐ igation images, it was determined that the tumor had reached a portion adjacent to the ambulatory motor area (green cross). D: The tumor was remained in this area (arrow). E: PDT at 150 mW/cm2 and 27 J/cm2 (diameter of the irradiation area: 1.5 cm2) was performed for tumor infiltration into this area. (aluminum foil protected the anterior cerebral arteries, arrow). F: Gadolinium-enhanced T1-weighted sagittal imaging on days 51 after surgery showed that

Photodynamic Therapy Using Talaporfin Sodium and Diode Laser for Newly Diagnosed Malignant Gliomas

http://dx.doi.org/10.5772/53485

269

gadolinium-enhancement at the site of PDT had disappeared.

Photodynamic Therapy Using Talaporfin Sodium and Diode Laser for Newly Diagnosed Malignant Gliomas http://dx.doi.org/10.5772/53485 269

and chemotherapy with oral TMZ. On day 3 after surgery, MRI showed remaining contrastenhanced tumor growth in the pre-motor area, but by the end of radiotherapy (on day 51 after surgery, Fig. 3F), complete response was confirmed at this site. His headache was dis‐

(a) (b)

(c)

appeared and he enjoyed a play of weekly golf until 3 years after operation.

268 Clinical Management and Evolving Novel Therapeutic Strategies for Patients with Brain Tumors

**Figure 3.** PDT and changes over time on postoperative magnetic resonance imaging (MRI) in Case 3. A: Preoperative axial FLAIR image showed the right frontal tumor and the peri-tumoral edema extending to the right pre-motor cor‐ tex. B: Preoperative gadolinium-enhanced T1-weighted sagittal imaging showed a heterogeneously enhanced tumor was present in the right superior frontal gyrus to supplementary motor cortex. C: Based on intra-operative optical nav‐ igation images, it was determined that the tumor had reached a portion adjacent to the ambulatory motor area (green cross). D: The tumor was remained in this area (arrow). E: PDT at 150 mW/cm2 and 27 J/cm2 (diameter of the irradiation area: 1.5 cm2) was performed for tumor infiltration into this area. (aluminum foil protected the anterior cerebral arteries, arrow). F: Gadolinium-enhanced T1-weighted sagittal imaging on days 51 after surgery showed that gadolinium-enhancement at the site of PDT had disappeared.

### **4.2. Summary**

In the evaluation of patients, of the 6 patients who were assessable after surgery, 3 achieved complete response, 2 achieved partial response, and 1 had progressive disease. The response rate was high at 83.3%. Regarding adverse events associated with PDT, brain edema involv‐ ing a large part of the middle cerebral artery perforator region occurred during PDT after insular glioma resection in Case 1. While it remains uncertain whether this was associated with the surgical procedure or PDT, the effect on normal capillary vessels should be closely monitored. No other obvious adverse events were observed. In most patients, postoperative chemo-radiation therapy was initiated, but recurrence was observed at the site of PDT in 7 patients. Despite recurrence, the progression-free survival time was 1 to 34 months (median: 22 months), and in cases receiving treatment after recurrence, 5 patients had a survival time of 6 to 14 months (median: 6 months). A further 3 patients of Grade 3 were alive during a follow-up period of 42 to 74 months (median: 50 months). An evaluation of 5 patients with glioblastoma also showed a median progression-free survival time of 14 months and a me‐ dian survival time of 26 months.

genation, and the depth from the site of laser irradiation. The irradiation laser factors included irradiation level and duration. How the cytotoxic effects can be enhanced while minimizing the vascular effects, and how ischemic damage to normal brain tissues can be minimized may well be the keys to determining whether or not PDT can be developed for

Photodynamic Therapy Using Talaporfin Sodium and Diode Laser for Newly Diagnosed Malignant Gliomas

mately 24 hours after the administration of TS. The practice of the PDT was performed on the diode laser (664nm) irradiation at a power density of 150 mW/cm2 for 180 seconds (27

Although there were only consecutive 8 patients of newly diagnosed malignant glioma in this series, PDT achieved a response rate of 83.3% and a median progression-free survival time of 14 months, a median survival time of 26 months in the glioblastoma patients who eventually died, and all 3 cases of Grade 3 patients were survived for 50 months median fol‐ low up period. In the 5 patients who achieved complete or partial response, preoperative KPS was improved. None of the patients had adverse events unequivocally attributable to PDT. The brain edema observed in Case 1 was possibly due to the surgical procedure, but the possibility of direct laser irradiation of brain blood vessels inducing vascular occlusion due to PDT could not be completely excluded. Therefore, normal blood vessels should be

**KPS after**

NE 60Gy ACNU/VCR/IFN 50 (+) 22 mon. Dead: 36 mon. CR 60Gy IFN 70 (+) 24 mon. Alive: 74 mon. CR 60Gy TMZ 90 (+) 34 mon. Dead: 40 mon. PR 60Gy TMZ 90 (+) 14 mon. Dead: 26 mon. PR 60Gy (-) 50 (+) 24 mon. Alive: 50 mon. PD (-) TMZ 40 (+) 1 mon. Dead: 7 mon. CR 60Gy PCZ/ACNU/VCR 80 (+) 6mon. Dead: 12mon. NE 60Gy TMZ 90 (-) Alive: 42mon.

KPS: Karnofsky Performance Scale, PDT: photodynamic therapy, RTx: radiation therapy, Chemo Tx: chemo‐ therapy, PFS: progression-free survival, Rt: right, Lt: left, Bil: bilateral, Anapl: anaplastic, CR: complete re‐ sponse, PR: partial response, PD: progression disease, NE: not evaluable, TMZ: temozolomide, VCR:

**Table 1.** Clinical summary of patients with newly diagnosed malignant glioma who received photodynamic therapy

**treatment Progression PFS Prognosis, Survival**

**period**

). We previously reported that this PDT protocol was considered safe in terms of the protection of early vascular endothelial cell damage and normal brain tissues, and could in‐

of TS and performed PDT approxi‐

http://dx.doi.org/10.5772/53485

271

the treatment of malignant glioma.

J/cm2

(PDT)

In the present study, we selected a dose of 40 mg/m2

duce the selective tumor cell damage by singlet oxygen [7].

covered with aluminum foil whenever possible.

vincristine, IFN: interferon-beta, PCZ: procarbazine, mon. months

**Response RTx Chemo Tx**

While it was possibly an adverse event associated with the surgical procedure, a causal rela‐ tionship with the PDT could not be excluded. In terms of photosensitivity, the photosensi‐ tive reaction resolved within 3 days after surgery (within 4 days after the intravenous injection) in all patients, at which point light shading at 500 lux was discontinued.

### **5. Discussion**

The objective of PDT is to selectively kill infiltrating tumor cells via a photochemical reac‐ tion, and this selectivity facilitates optimal local control therapy [2], making it possible to maintain brain function. PDT is designed to cause damage at the cellular level in the form of apoptosis [2]. There are numerous studies which have reported that the main mechanism of PDT is the induction of vascular occlusion from fibrin thrombus formation due to vascular endothelial cell damage [5]. In particular, it has been assumed that the main mechanism on cellular damage induced by PDT using TS in conjunction with a diode laser irradiation is indirect damage in the form of coagulation necrosis by tumor vascular occlusion [5]. In our basic experimental study, C6 glioma tissue showed coagulation necrosis in tissues located in a portion near the tumor surface that was laser irradiated, and cellular death in the distal portion [3]. However, in the actual area of tissue damage, 80% of the area was considered to be tissue damage caused by the coagulation necrosis. Yamamoto et al. [5] reported that fac‐ tor XIII was activated by vascular endothelial cell damage within 20 seconds following PDT of cancer tissue. A fibrin thrombus formed as a consequence, which appeared to be the main mechanism of PDT.

Our strategy was to optimally reduce vascular damage caused by PDT, and to ensure safety to avoid ischemic damage to normal brain tissues. Madsen et al. [6] reported that the factors which influence the effects of PDT were tissue factors and irradiation laser factors. The tis‐ sue factors included the tissue concentrations of the photosensitizer, the level of tissue oxy‐ genation, and the depth from the site of laser irradiation. The irradiation laser factors included irradiation level and duration. How the cytotoxic effects can be enhanced while minimizing the vascular effects, and how ischemic damage to normal brain tissues can be minimized may well be the keys to determining whether or not PDT can be developed for the treatment of malignant glioma.

**4.2. Summary**

dian survival time of 26 months.

**5. Discussion**

mechanism of PDT.

In the evaluation of patients, of the 6 patients who were assessable after surgery, 3 achieved complete response, 2 achieved partial response, and 1 had progressive disease. The response rate was high at 83.3%. Regarding adverse events associated with PDT, brain edema involv‐ ing a large part of the middle cerebral artery perforator region occurred during PDT after insular glioma resection in Case 1. While it remains uncertain whether this was associated with the surgical procedure or PDT, the effect on normal capillary vessels should be closely monitored. No other obvious adverse events were observed. In most patients, postoperative chemo-radiation therapy was initiated, but recurrence was observed at the site of PDT in 7 patients. Despite recurrence, the progression-free survival time was 1 to 34 months (median: 22 months), and in cases receiving treatment after recurrence, 5 patients had a survival time of 6 to 14 months (median: 6 months). A further 3 patients of Grade 3 were alive during a follow-up period of 42 to 74 months (median: 50 months). An evaluation of 5 patients with glioblastoma also showed a median progression-free survival time of 14 months and a me‐

270 Clinical Management and Evolving Novel Therapeutic Strategies for Patients with Brain Tumors

While it was possibly an adverse event associated with the surgical procedure, a causal rela‐ tionship with the PDT could not be excluded. In terms of photosensitivity, the photosensi‐ tive reaction resolved within 3 days after surgery (within 4 days after the intravenous

The objective of PDT is to selectively kill infiltrating tumor cells via a photochemical reac‐ tion, and this selectivity facilitates optimal local control therapy [2], making it possible to maintain brain function. PDT is designed to cause damage at the cellular level in the form of apoptosis [2]. There are numerous studies which have reported that the main mechanism of PDT is the induction of vascular occlusion from fibrin thrombus formation due to vascular endothelial cell damage [5]. In particular, it has been assumed that the main mechanism on cellular damage induced by PDT using TS in conjunction with a diode laser irradiation is indirect damage in the form of coagulation necrosis by tumor vascular occlusion [5]. In our basic experimental study, C6 glioma tissue showed coagulation necrosis in tissues located in a portion near the tumor surface that was laser irradiated, and cellular death in the distal portion [3]. However, in the actual area of tissue damage, 80% of the area was considered to be tissue damage caused by the coagulation necrosis. Yamamoto et al. [5] reported that fac‐ tor XIII was activated by vascular endothelial cell damage within 20 seconds following PDT of cancer tissue. A fibrin thrombus formed as a consequence, which appeared to be the main

Our strategy was to optimally reduce vascular damage caused by PDT, and to ensure safety to avoid ischemic damage to normal brain tissues. Madsen et al. [6] reported that the factors which influence the effects of PDT were tissue factors and irradiation laser factors. The tis‐ sue factors included the tissue concentrations of the photosensitizer, the level of tissue oxy‐

injection) in all patients, at which point light shading at 500 lux was discontinued.

In the present study, we selected a dose of 40 mg/m2 of TS and performed PDT approxi‐ mately 24 hours after the administration of TS. The practice of the PDT was performed on the diode laser (664nm) irradiation at a power density of 150 mW/cm2 for 180 seconds (27 J/cm2 ). We previously reported that this PDT protocol was considered safe in terms of the protection of early vascular endothelial cell damage and normal brain tissues, and could in‐ duce the selective tumor cell damage by singlet oxygen [7].

Although there were only consecutive 8 patients of newly diagnosed malignant glioma in this series, PDT achieved a response rate of 83.3% and a median progression-free survival time of 14 months, a median survival time of 26 months in the glioblastoma patients who eventually died, and all 3 cases of Grade 3 patients were survived for 50 months median fol‐ low up period. In the 5 patients who achieved complete or partial response, preoperative KPS was improved. None of the patients had adverse events unequivocally attributable to PDT. The brain edema observed in Case 1 was possibly due to the surgical procedure, but the possibility of direct laser irradiation of brain blood vessels inducing vascular occlusion due to PDT could not be completely excluded. Therefore, normal blood vessels should be covered with aluminum foil whenever possible.

KPS: Karnofsky Performance Scale, PDT: photodynamic therapy, RTx: radiation therapy, Chemo Tx: chemo‐ therapy, PFS: progression-free survival, Rt: right, Lt: left, Bil: bilateral, Anapl: anaplastic, CR: complete re‐ sponse, PR: partial response, PD: progression disease, NE: not evaluable, TMZ: temozolomide, VCR: vincristine, IFN: interferon-beta, PCZ: procarbazine, mon. months

**Table 1.** Clinical summary of patients with newly diagnosed malignant glioma who received photodynamic therapy (PDT)

The present study of PDT using talaporfin sodium in conjunction with diode laser irradiation for malignant glioma supports the validity of PDT for malignant glioma in Japan. Laser irra‐ diation used to activate TS has a longer wavelength (664 nm) than that used with other photo‐ sensitizers, such as Porfimer sodium [8-11], 5-ALA [12-14] and m-THPC [15, 16], and theoretically, it can penetrate brain tissue to a greater depth. We believe that our strategy will demonstrate a therapeutic effect not to be inferior to those of previous reports [17] using other photosensitizers. In future, it is necessary to perform the larger-scale prospective clinical study to confirm the clinical feasibility of this novel therapeutic option to malignant gliomas.

[4] Kato H, Furukawa K, Sato M, Okunaka T, Kusunoki Y, Kawahara M, Fukuoka M, Miyazawa T, Yana T, Matsui K, Shiraishi T, Horinouchi H. Phase II clinical study of photodynamic therapy using mono-L-aspartyl chlorine e6 and diode laser for early

Photodynamic Therapy Using Talaporfin Sodium and Diode Laser for Newly Diagnosed Malignant Gliomas

http://dx.doi.org/10.5772/53485

273

[5] Yamamoto Y, Shibuya H, Okunaka T, Aizawa K, Kato H. Fibrin plugging as a cause of microcirculatory occlusion during photodynamic therapy. Lasers Med Sci 1999,

[6] Madsen SJ, Angell-Petersen E, Spetalen S, Carper SW, Ziegler SA, Hirschberg H. Photodynamic therapy of newly implanted glioma cells in the rat brain. Lasers Surg

[7] Akimoto J, Haraoka J, Aizawa K. Preliminary clinical report on safety and efficacy of photodynamic therapy using Talaporfin sodium for malignant gliomas. Photodiag

[8] Muller PJ, Wilson BC. Photodynamic therapy for malignant newly diagnosed supra‐

[9] Muller PJ, Wilson BC. Photodynamic therapy for recurrent supratentorial gliomas.

[10] Popovic EA, Kaye AH, Hill JS. Photodynamic therapy of brain tumors. Semin Surg

[11] Stylli SS, Kaye AH, MacGregor L, Howes M, Rajendra P. Photodynamic therapy of high grade glioma - long term survival. J Clin Neurosci 2005, 12(4), 389-398

[12] Beck TJ, Kreth FW, Beyer W, Mehrkens JH, Obermeier A, Stepp H, Stummer W, Baumgartner R. Interstitial photodynamic therapy of nonresectable malignant glio‐ ma recurrences using 5-aminolevulinic acid induced protoporphyrin IX. Lasers Surg

[13] Stummer W, Beck T, Beyer W, Mehrkens JH, Obermeier A, Etminan N, Stepp H, Tonn JC, Baumgartner R, Herms J, Kreth FW. Long-sustaining response in a patient with non-resectable, distant recurrence of glioblastoma multiforme treated by inter‐ stitial photodynamic therapy using 5-ALA: case report. J Neurooncol 2008,

[14] Bisland SK, Ligle A, Lin A,Rusnow R, Wilson BC. Metronomic photodynamic thera‐ py as a new paradigm for photodynamic therapy: rationale and preclinical evalua‐ tion of technical feasibility for treating malignant brain tumors.  Photochem Photobiol 2001, 74(4), 611-616

a phase I/II trial. Br J Neurosurg 1988, 2(2), 241-248

tentorial gliomas. J Clin Laser Med Surg 1996, 14, 263-270

squamous cell carcinoma of the lung. Lung Cancer 2003, 42, 103-111

14,129-135

Med 2006, 38, 540-548

Oncol 1995, 11, 335-345

Med 2007, 39, 386-393

87,103-109

2004, 80, 22-30

Photodyn Therapy 2012, 9, 91-99

Semin Surg Oncol 1996, 14, 263-270

### **6. Conclusions**

We performed PDT using TS in conjunction with diode laser irradiation in areas of tumor invasion, following resection of adult malignant glioma which had infiltrated the eloquent areas. We selected a 24 hours interval after the administration of TS before commencing PDT and reduced irradiation energy intensity of the diode laser to 27 J/cm2 to preserve nor‐ mal brain function. The therapy achieved with no adversed event directly attributable to PDT and a response rate of 83.3% in patients with a newly diagnosed malignant gliomas.

### **Author details**

Jiro Akimoto

Address all correspondence to<EMAIL_ADDRESS>
Department of Neurosurgery, Tokyo Medical University, Tokyo, Japan

The authors report no conflict of interest concerning the materials or methods used in this study and findings specified in this paper.

### **References**

[4] Kato H, Furukawa K, Sato M, Okunaka T, Kusunoki Y, Kawahara M, Fukuoka M, Miyazawa T, Yana T, Matsui K, Shiraishi T, Horinouchi H. Phase II clinical study of photodynamic therapy using mono-L-aspartyl chlorine e6 and diode laser for early squamous cell carcinoma of the lung. Lung Cancer 2003, 42, 103-111

The present study of PDT using talaporfin sodium in conjunction with diode laser irradiation for malignant glioma supports the validity of PDT for malignant glioma in Japan. Laser irra‐ diation used to activate TS has a longer wavelength (664 nm) than that used with other photo‐ sensitizers, such as Porfimer sodium [8-11], 5-ALA [12-14] and m-THPC [15, 16], and theoretically, it can penetrate brain tissue to a greater depth. We believe that our strategy will demonstrate a therapeutic effect not to be inferior to those of previous reports [17] using other photosensitizers. In future, it is necessary to perform the larger-scale prospective clinical study to confirm the clinical feasibility of this novel therapeutic option to malignant gliomas.

272 Clinical Management and Evolving Novel Therapeutic Strategies for Patients with Brain Tumors

We performed PDT using TS in conjunction with diode laser irradiation in areas of tumor invasion, following resection of adult malignant glioma which had infiltrated the eloquent areas. We selected a 24 hours interval after the administration of TS before commencing

mal brain function. The therapy achieved with no adversed event directly attributable to PDT and a response rate of 83.3% in patients with a newly diagnosed malignant gliomas.

The authors report no conflict of interest concerning the materials or methods used in this

[1] Wilson CB. Glioblastoma: the past, the present, and the future. Clin Neurosurg 1992,

[2] Castano AP, Demidova TN, Hambrin MR. Mechanism of photodynamic therapy: Part three - Photosensitizer pharmacokinetics, biodistribution, tumor localization and

[3] Namatame H, Akimoto J, Matsumura H, Haraoka J, Aizawa K. Photodynamic thera‐ py of C6 implanted glioma cells in the rat brain employing Talaporfin sodium. Pho‐

modes of tumor destruction. Photodiag Photodyn Therapy 2005, 2(2), 91-106

to preserve nor‐

PDT and reduced irradiation energy intensity of the diode laser to 27 J/cm2

Address all correspondence to<EMAIL_ADDRESS>
todiag Photodyn Therapy 2008, 5, 198-209

study and findings specified in this paper.

Department of Neurosurgery, Tokyo Medical University, Tokyo, Japan

**6. Conclusions**

**Author details**

Jiro Akimoto

**References**

38, 32-48

[17] Eljamel MS, Goodman C, Moseley H. ALA and Photofrin fluorescence-guided resec‐ tion and repetitive PDT in glioblastoma multiforme: a single centre Phase III rando‐ mised controlled trial. Lasers Med Sci 2008, 23(4), 361-7

**Section 6**

**Molecular Biology of Brain Tumors**

**Molecular Biology of Brain Tumors**

[17] Eljamel MS, Goodman C, Moseley H. ALA and Photofrin fluorescence-guided resec‐ tion and repetitive PDT in glioblastoma multiforme: a single centre Phase III rando‐

mised controlled trial. Lasers Med Sci 2008, 23(4), 361-7

274 Clinical Management and Evolving Novel Therapeutic Strategies for Patients with Brain Tumors

**Chapter 14**

**Epithelial to Mesenchymal Transition and Progression of**

Glioblastoma multiforme (GBM) is the most common primary brain tumor among adults. Rapid tumor progression and diffuse invasion of brain tissue restrict the therapeutic options and result in poor prognosis despite advances in the understanding of this tumor's molecular biology and pathophysiology [1-4]. Current standard therapy consists of a combination of tumor resection, irradiation and temozolomide. Advances in the field of molecular biology have led to the development of targeted therapy, and the epidermal growth factor receptor (EGFR) has been introduced as one potential therapeutic target [5]. Although pre-clinical studies have shown promising effects, clinical applications yielded no significant benefit in comparison with standard therapy. This fact encouraged extensive investigations studying the molecular mechanisms underlying GBM resistance to EGFR-targeted therapy. Epithelial to mesenchymal transition (EMT) is considered an important factor contributing to resistance

Epithelial to mesenchymal transition is a common process, taking part in organ development, wound healing, tissue remodelling, cancer progression and metastasis. The main features accompanying this mechanism are the loss of epithelial characteristics of cells and the acquis‐ ition of mesenchymal markers such as fibronectin, vimentin and N-cadherin. As a result, tumor cells develop increasing invasive and migratory potential. The loss of epithelial features mounts the resistance of tumor cells against targeted therapy directed towards the EGFR. Mesenchymal transition leads to disorganisation of the cytoskeleton, disruption of intercellu‐ lar adhesions and changes in expression of transcriptional factors, resulting in the development

> © 2013 Pala et al.; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use,

© 2013 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution,

distribution, and reproduction in any medium, provided the original work is properly cited.

and reproduction in any medium, provided the original work is properly cited.

**Glioblastoma**

Andrej Pala, Georg Karpel-Massler,

http://dx.doi.org/10.5772/53183

**1. Introduction**

Christian Rainer Wirtz and Marc-Eric Halatsch

Additional information is available at the end of the chapter

towards this therapy by diminishing the molecular target [1-6].

of a more malignant cellular phenotype [1, 2, 7].

## **Epithelial to Mesenchymal Transition and Progression of Glioblastoma**

Andrej Pala, Georg Karpel-Massler, Christian Rainer Wirtz and Marc-Eric Halatsch

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/53183

### **1. Introduction**

Glioblastoma multiforme (GBM) is the most common primary brain tumor among adults. Rapid tumor progression and diffuse invasion of brain tissue restrict the therapeutic options and result in poor prognosis despite advances in the understanding of this tumor's molecular biology and pathophysiology [1-4]. Current standard therapy consists of a combination of tumor resection, irradiation and temozolomide. Advances in the field of molecular biology have led to the development of targeted therapy, and the epidermal growth factor receptor (EGFR) has been introduced as one potential therapeutic target [5]. Although pre-clinical studies have shown promising effects, clinical applications yielded no significant benefit in comparison with standard therapy. This fact encouraged extensive investigations studying the molecular mechanisms underlying GBM resistance to EGFR-targeted therapy. Epithelial to mesenchymal transition (EMT) is considered an important factor contributing to resistance towards this therapy by diminishing the molecular target [1-6].

Epithelial to mesenchymal transition is a common process, taking part in organ development, wound healing, tissue remodelling, cancer progression and metastasis. The main features accompanying this mechanism are the loss of epithelial characteristics of cells and the acquis‐ ition of mesenchymal markers such as fibronectin, vimentin and N-cadherin. As a result, tumor cells develop increasing invasive and migratory potential. The loss of epithelial features mounts the resistance of tumor cells against targeted therapy directed towards the EGFR. Mesenchymal transition leads to disorganisation of the cytoskeleton, disruption of intercellu‐ lar adhesions and changes in expression of transcriptional factors, resulting in the development of a more malignant cellular phenotype [1, 2, 7].

© 2013 Pala et al.; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2013 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

In embryogenesis, epithelial to mesenchymal transition appears during gastrulation and activates the formation of the mesodermal layer as the third germ layer after ectoderm and endoderm are built. In addition, EMT is involved in the development of neural crest cells, arising from the dorsal part of the neural tube [7]. This requires a highly specialised program that is necessary to allow the normal development of cells. On the other hand, under patho‐ logical conditions, EMT increases the invasiveness and migratory potential of tumor cells, supports their malignant transformation and may lead to resistance of tumor cells towards various therapeutic agents, especially in malignant gliomas.

Epidermal growth factor overexpression and/or mutational activation is the most common molecular alteration in primary GBM and can be detected in far more than 50% of glioblastoma cells [5]. There are several mechanisms that may cause dysfunction of the receptor. Epidermal growth factor receptor gene amplification is the most common reason leading to protein overexpression. Other mechanisms include constitutive receptor activation by deletionmutation (EGFRvIII) or autocrine overproduction of EGFR ligands [5, 6, 9]. The presence of

Epithelial to Mesenchymal Transition and Progression of Glioblastoma

http://dx.doi.org/10.5772/53183

279

Several drugs that target EGFR have been introduced. The TK inhibitors erlotinib and gefitinib compete with adenosine triphosphate to bind to the intracellular TK domain and dissolve further signalling involving PI3-K/AKT and MAPK pathways. Erlotinib was approved in the treatment of metastatic non-small cell lung carcinoma (NSCLC) and in combination with gemcitabine for metastasized pancreatic cancer or pancreatic cancer without the possibility of gross total resection [11, 12]. Gefitinib is used for metastatic NSCLC. Another small molecule EGFR-TK inhibitor represents lapatinib, which affords dual inhibition of EGFR and HER-2 and has been approved for the treatment of advanced or metastatic breast cancer. Unfortu‐ nately, the clinical application in gliomas had shown no benefit in comparison to standard

Another possibility to inhibit EGFR signalling consists in the prevention of ligand binding. Monoclonal antibodies such as cetuximab have been introduced and investigated for this purpose. However, their molecular weight impairs their penetration of the blood brain barrier. Often the concentration of the agent is insufficient to yield a significant therapeutic effect

Antisense RNA has been introduced as an option to inhibit the EGFR pathway as well. Sense RNA hybridizes to antisense RNA which leads to inhibition of translation. Another strategy to switch off the EGFR cascade is the application of small interfering RNAs. These suppress the homolog genes and result in sequence-specific degradation of mRNA. Furthermore, ribozymes are small RNA molecules that are able to bind and cleave complementary RNA substrates. EGFRvIII-targeted ribozymes have been reported to reverse malignant phenotype of transformed fibroblasts and glioblastoma cells [14, 15]. However, the potential clinical value

Tumor progression represents an integrated multistep complex of molecular changes that ultimately result in local invasion and dissemination of malignant cells. The cells invade the epithelial basement membrane, infiltrate the surrounding tissue, access the blood circu‐ lation and may create new tumor seeds. This dynamic and aggressive process demands

EGFRvIII is associated with EGFR gene amplification.

**3. Therapeutic EGFR inhibitors**

therapy [1, 5, 6, 8].

within the targeted tissue [13].

of these methods remains unknown [16].

**4. Molecular principles of EMT**

### **2. EGFR**

The EGFR is a transmembraneous glycoprotein belonging to the human EGFR (HER) family, which includes four members (HER 1-4) that share a similar structure. The EGFR/HER1 consists of external and internal domains. The extracellular part possesses a binding sequence for ligands such as EGF, hepatocyte growth factor (HGF), fibroblast growth factor (FGF), insulin-like growth factor-1 (IGF-1) or tumor growth factor-α (TGF-α). The internal domain contains a tyrosine kinase (TK), capable of phosphorylation and activation of a variety of downstream pathways such as phospholipase Cγ, phosphatidylinositol-3-kinase (PI3-K), Janus-activated kinase 2 (JAK-2) and mitogen-activated kinase (MAPK). Human epidermal growth factor family receptors are expressed on many cell types and mediate signal transduc‐ tion to intracellular compartments and hereby regulate various functions of cells such as proliferation or differentiation [1, 5, 8, 9].

Phosphorylation and dephosphorylation are crucial pathophysiological events within intracellular interactions. A dysregulation of their balance contributes to the development of an invasive cellular phenotype. Upon activation, receptor tyrosine kinases dimerise and autophosphorylate on tyrosine residues, which in turn create complexes with proteins possessing SH2 domains such as GRB2, SHC or signalling proteins PI3-K and SRC [9].

Pathological activation of RAS occurs in numerous cancers. This signalling pathway inte‐ grates various diverse stimuli promoting cell transformation, gene expression and tumor progression. RAS is a small guanine triphosphate-binding protein that activates the serinethreonine kinase RAF. RAF phosphorylates the dual specificity kinase MEK, which in turn activates MAPK (ERK). The latter translocate into the nucleus and activate various tran‐ scription factors such as the ETS family factors FOS, JUN or SLUG (SNAI-2). Thereby RAS propagates the activation of EMT. Moreover, RAS is able to promote EMT by downstream activation of PI3-K or RHO/RAC. PI3-K is involved in cell cycle regulation via phosphory‐ lation of cyclin-dependent kinase inhibitors. In addition, it controls the phosphorylation of proapoptotic proteins and survival of the cell. PI3-K can be activated either via RAS or di‐ rectly via TK receptors. PI3-K is a lipide kinase, and its activation generates phosphatidyli‐ nositol-3,4,5-triphosphate (PIP3) at the inner side of plasma membrane, which furthermore mediates the signalling via the serine-threonine kinase AKT that is able to translocate into the nucleus and take part in gene regulation through phosphatidylinositol-dependent pro‐ tein kinase-1 (PDK-1) [8, 9, 10].

Epidermal growth factor overexpression and/or mutational activation is the most common molecular alteration in primary GBM and can be detected in far more than 50% of glioblastoma cells [5]. There are several mechanisms that may cause dysfunction of the receptor. Epidermal growth factor receptor gene amplification is the most common reason leading to protein overexpression. Other mechanisms include constitutive receptor activation by deletionmutation (EGFRvIII) or autocrine overproduction of EGFR ligands [5, 6, 9]. The presence of EGFRvIII is associated with EGFR gene amplification.

### **3. Therapeutic EGFR inhibitors**

In embryogenesis, epithelial to mesenchymal transition appears during gastrulation and activates the formation of the mesodermal layer as the third germ layer after ectoderm and endoderm are built. In addition, EMT is involved in the development of neural crest cells, arising from the dorsal part of the neural tube [7]. This requires a highly specialised program that is necessary to allow the normal development of cells. On the other hand, under patho‐ logical conditions, EMT increases the invasiveness and migratory potential of tumor cells, supports their malignant transformation and may lead to resistance of tumor cells towards

The EGFR is a transmembraneous glycoprotein belonging to the human EGFR (HER) family, which includes four members (HER 1-4) that share a similar structure. The EGFR/HER1 consists of external and internal domains. The extracellular part possesses a binding sequence for ligands such as EGF, hepatocyte growth factor (HGF), fibroblast growth factor (FGF), insulin-like growth factor-1 (IGF-1) or tumor growth factor-α (TGF-α). The internal domain contains a tyrosine kinase (TK), capable of phosphorylation and activation of a variety of downstream pathways such as phospholipase Cγ, phosphatidylinositol-3-kinase (PI3-K), Janus-activated kinase 2 (JAK-2) and mitogen-activated kinase (MAPK). Human epidermal growth factor family receptors are expressed on many cell types and mediate signal transduc‐ tion to intracellular compartments and hereby regulate various functions of cells such as

Phosphorylation and dephosphorylation are crucial pathophysiological events within intracellular interactions. A dysregulation of their balance contributes to the development of an invasive cellular phenotype. Upon activation, receptor tyrosine kinases dimerise and autophosphorylate on tyrosine residues, which in turn create complexes with proteins possessing SH2 domains such as GRB2, SHC or signalling proteins PI3-K and SRC [9].

Pathological activation of RAS occurs in numerous cancers. This signalling pathway inte‐ grates various diverse stimuli promoting cell transformation, gene expression and tumor progression. RAS is a small guanine triphosphate-binding protein that activates the serinethreonine kinase RAF. RAF phosphorylates the dual specificity kinase MEK, which in turn activates MAPK (ERK). The latter translocate into the nucleus and activate various tran‐ scription factors such as the ETS family factors FOS, JUN or SLUG (SNAI-2). Thereby RAS propagates the activation of EMT. Moreover, RAS is able to promote EMT by downstream activation of PI3-K or RHO/RAC. PI3-K is involved in cell cycle regulation via phosphory‐ lation of cyclin-dependent kinase inhibitors. In addition, it controls the phosphorylation of proapoptotic proteins and survival of the cell. PI3-K can be activated either via RAS or di‐ rectly via TK receptors. PI3-K is a lipide kinase, and its activation generates phosphatidyli‐ nositol-3,4,5-triphosphate (PIP3) at the inner side of plasma membrane, which furthermore mediates the signalling via the serine-threonine kinase AKT that is able to translocate into the nucleus and take part in gene regulation through phosphatidylinositol-dependent pro‐

various therapeutic agents, especially in malignant gliomas.

278 Clinical Management and Evolving Novel Therapeutic Strategies for Patients with Brain Tumors

proliferation or differentiation [1, 5, 8, 9].

tein kinase-1 (PDK-1) [8, 9, 10].

**2. EGFR**

Several drugs that target EGFR have been introduced. The TK inhibitors erlotinib and gefitinib compete with adenosine triphosphate to bind to the intracellular TK domain and dissolve further signalling involving PI3-K/AKT and MAPK pathways. Erlotinib was approved in the treatment of metastatic non-small cell lung carcinoma (NSCLC) and in combination with gemcitabine for metastasized pancreatic cancer or pancreatic cancer without the possibility of gross total resection [11, 12]. Gefitinib is used for metastatic NSCLC. Another small molecule EGFR-TK inhibitor represents lapatinib, which affords dual inhibition of EGFR and HER-2 and has been approved for the treatment of advanced or metastatic breast cancer. Unfortu‐ nately, the clinical application in gliomas had shown no benefit in comparison to standard therapy [1, 5, 6, 8].

Another possibility to inhibit EGFR signalling consists in the prevention of ligand binding. Monoclonal antibodies such as cetuximab have been introduced and investigated for this purpose. However, their molecular weight impairs their penetration of the blood brain barrier. Often the concentration of the agent is insufficient to yield a significant therapeutic effect within the targeted tissue [13].

Antisense RNA has been introduced as an option to inhibit the EGFR pathway as well. Sense RNA hybridizes to antisense RNA which leads to inhibition of translation. Another strategy to switch off the EGFR cascade is the application of small interfering RNAs. These suppress the homolog genes and result in sequence-specific degradation of mRNA. Furthermore, ribozymes are small RNA molecules that are able to bind and cleave complementary RNA substrates. EGFRvIII-targeted ribozymes have been reported to reverse malignant phenotype of transformed fibroblasts and glioblastoma cells [14, 15]. However, the potential clinical value of these methods remains unknown [16].

### **4. Molecular principles of EMT**

Tumor progression represents an integrated multistep complex of molecular changes that ultimately result in local invasion and dissemination of malignant cells. The cells invade the epithelial basement membrane, infiltrate the surrounding tissue, access the blood circu‐ lation and may create new tumor seeds. This dynamic and aggressive process demands cell-to-cell and cell-to-matrix interactions, degradation and remodelling of extracellular ma‐ trix, cytoskeleton reorganisation and gain of migratory behaviour of tumor cells. Epithelial to mesenchymal transition is a differentiation switch which promotes the loss of the epithe‐ lial phenotype and facilitates cell motility and invasiveness. One of the crucial steps pro‐ moting EMT is the repression of the epithelial marker E-cadherin, a transmembraneous calcium-dependent glycoprotein responsible for cell-cell adhesion, which plays a key role in embryonic development and homeostasis. The intracellular domain of E-cadherin asso‐ ciates with a protein complex involving α-catenin, β-catenin and p120-catenin. Actin pro‐ teins bind α-catenin and hereby allow the connection between E-cadherin and the actin microfilament network of the cytoskeleton that forms the shape of the cell. Epithelial cells are typically characterized by apicobasal difference. Thus, the apical compartment faces the lumen, whereas the basolateral surface is located on a basement membrane. Epithelial cells are anchored by specialized cell-to-cell connections, i.e. tight junctions, adherens junction and desmosomes. E-cadherin acts as a tumor suppressor by linking the epithelial cells and keeping them in a stationary rigid state [1, 2, 7, 8, 9, 17].

apoptosis, TWIST-1 enables cell survival by preventing growth arrest and promotes invasive‐ ness of tumor cells [3, 4]. Moreover, this process may result in increased resistance to various

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281

Sex determining region Y-box-2, also known as SOX-2, is another transcription factor that is co-expressed with TWIST-1, playing an important role during the development of the nervous system and tumor progression. Knockdown of SOX-2 results in reduced expression of TWIST-1 [4], promoting EMT and potentially being essential for tumor progression and the develop‐