**The Cytoprotective Effect of Amifostine Against Radiation Induced Toxicity**

Vassilis E. Kouloulias1 and John R. Kouvaris2 *1Attikon Hospital University of Athens, Medical School, 2Aretaieion Hospital University of Athens, Medical School, Athens, Greece* 

#### **1. Introduction**

256 Modern Practices in Radiation Therapy

RoswitB, Malsky SJ, Reid CB. Severe radiation injuries of the stomach, small intestine, colon and rectum. Am J Roentgenol Radium Ther Nucl Med 1972; 114:460-75. Chen KY, Withers HR. Survival characteristics of stem cells of gastric mucosa in C 3 H mice

Ajani JA, Winter K, Okawara GS, et al. Phase II trial of preoperative chemoradiation in

Otsuka T, Noda T, Yokoo M, Ibaraki K. Recurrent gastric perforation as a late complication

Coia LR, Myerson RJ, Tepper JE. Late effects of radiation therapy on the gastrointestinal

Grigsby PW, Heydon K, Mutch DG, Kim RY, Eifel P. Long-term follow-up of RTOG 92-10:

Griem MI, Kleinerman RA, Boice JD, Stowall M, Shefner D, Lubin JH. Cancer following

Cosset JM, Henry-Amar M, Burgers JM, et al. Late radiation injuries of the gastrointestinal

Yang MH, Lee JH, Choi MS, et al. Gastrointestinal complications after radiation therapy in patients with hepatocellular carcinoma. Hepatogastroenterology 2005;52:1759-63. Goldstein H, Rogers L, Fletcher G, Dodd G. Radiological manifestations of radiation-induced injury to the normal upper gastrointestinal tract. Radiology 1975;117:135-40. Hoyer M, Roed H, Sengelov L, et al. Phase-II study on stereotactic radiotherapy of locally

European Society for Therapeutic Radiology and Oncology 2005;76:48-53. Murphy JD, Christman-Skieller C, Kim j, Dieterich s, Chang DT. A Dosimetric Model of

Lee MT, Kim JJ, Dinniwell R, etal.Phase I study of individualized stereotactic body

Streitparth F, Pech M, Böhmig M, et al. In vivo assessment of the gastric mucosal tolerance

Emami B, Lyman J, Brown A, et al. Tolerance of normal tissue to therapeutic irradiation. Int

Matzinger O, Gerber E, Bernstein Z, Maingon P, Haustermans K, Bosset JF, Gulyban A,

radiotherapy of liver metastases. J Clin Oncol 2009; 27:1585–1591.

exploratory laparotomy and fractionation. Radiother Oncol 1988;13:61-8. Mohiuddin M, Rosato F, Barbot D, Schricht A, Biermann W, Cantor R. Long term results of

radiotherapy for peptic ulcer. J Natl Cancer Inst 1994 ;86 :842-9.

modality therapy and pathologic response. J Clin Oncol 2006;24:3953-8. Busch DB. Radiation and chemotherapy injury: pathophysiology, diagnosis, and treatment.

Med 1972;21:521-34.

Crit Rev Oncol Hematol 1993;15:49-89.

tract. Int J Radiat Oncol Biol Phys 1995;31:1213-36.

pancreas. Int J RadiatOncol Biol Phys 1992;23:305\_11.

J Radiat Oncol Biol Phys, 2009;78:1420-1426a

J Radiat Oncol Biol Phys 1991;21:109-22.

Aug;92(2):164-75. Epub 2009 Apr 15.

Biol Phys 2006;65:1479-86.

Intern Med 2008;47:1407-9.

2001;51:982-7.

subjected to localized gamma irradiation. Int J Radiat Biol Relat Stud Phys Chem

patients with localized gastric adenocarcinoma (RTOG 9904): quality of combined

of radiotherapy for mucosa-associated lymphoid tissue lymphoma of the stomach.

cervical cancer with positive para-aortic lymph nodes. Int J Radiat Oncol Biol Phys

tract in the H2 and H5 EORTC Hodgkin's disease trials: emphasis on the role of

combined modality treatment with I-125implantation for carcinoma of the

advanced pancreatic carcinoma. Radiotherapy and oncology : journal of the

Duodenal Toxicity After Stereotactic Body Radiotherapy for Pancreatic Cancer. Int

dose after single fraction, small volume irradiation of liver malignancies by computed tomography-guided, high-dose-rate brachytherapy. Int J Radiat Oncol

Poortmans P, Collette L, Kuten A.EORTC-ROG expert opinion: radiotherapy volume and treatment guidelines for neoadjuvant radiation of adenocarcinomas of the gastroesophageal junction and the stomach. Radiother Oncol. 2009 Radiation treatment is an important therapeutic option for a number of malignancies (American Cancer Society), but its use is frequently limited by adverse effects on normal tissues (Stone et al., 2003). Thus, the goal of most oncology treatments is to maximize the antineoplastic effect while minimizing deleterious outcomes for the patient. WR-2721 was developed by the U.S. Army Anti-Radiation Drug Development Program for its potential to protect against damage caused by ionizing radiation (Yuhas &Stoner, 1969). Today, WR-2721 is known as amifostine (Ethyol®; MedImmune Oncology, Inc., Gaithersburg, MD). Initial preclinical studies demonstrated that amifostine could protect treated mice from lethal doses of radiation, and this protection did not extend to transplanted mammary tumor cells (Yuhas &Stoner, 1969).

Amifostine, a thiol that protects cells from damage by scavenging oxygen-derived free radicals, was later evaluated for a potential role in reducing the toxicities from radiation and chemotherapeutic agents, such as alkylating agents and platinum agents. In contrast to organspecific protectants, amifostine is considered a broad-spectrum cytoprotective agent (Hensley et al., 1999). Preclinical studies demonstrated that amifostine can selectively protect almost all normal tissues from the cytotoxic effects of some chemotherapeutic agents and radiation therapy. Neoplastic tissues do not benefit from amifostine's protection (Koukourakis, 2003; Sasse et al., 2006; Yuhas et al., 1980). Amifostine is an inactive prodrug that cannot protect until dephosphorylated to the active metabolite, WR-1065, by alkaline phosphatase in the plasma membrane (Calabro-Jones et al., 1985). The selective protection of normal tissue is the result of a greater accumulation of WR-1065 in normal tissues than in tumor cells. Tumors are relatively hypovascular, thus resulting in comparative hypoxia and a low interstitial pH. Furthermore, alkaline phosphatase expression is reduced in malignant tissues. Taken together, the combination of hypovascularity, low pH, and reduced enzyme levels results in low accumulation of active drug in tumor tissues. Thus, normal tissues may be able to maintain as much as a 100-fold greater concentration of the free thiol than tumor tissue (Yuhas, 1980).

Once inside the cell, WR-1065 scavenges free radicals, protecting cellular membranes and DNA from damage. However, other studies have suggested that additional mechanisms may also play important roles in the action of amifostine. In vitro studies have shown that

The Cytoprotective Effect of Amifostine Against Radiation Induced Toxicity 259

xerostomia or grade 3 oral mucositis between patients receiving i.v. amifostine and those receiving placebo (Buentzel et al., 2006). On days when combined radiochemotherapy was administered, timing between amifostine and radiotherapy may have exceeded 60 minutes. The authors suggest that timing of the amifostine doses relative to the beginning of radiotherapy may have influenced efficacy because of inadequate exposure to amifostine. In addition, the observed rates of grade 2 acute xerostomia and grade 3 oral mucositis in the placebo group were unexpectedly low, reducing the ability of the study to show significant benefit with amifostine. In contrast, studies in which amifostine was administered within 30 minutes of radiotherapy have shown promise with regard to protection from acute and chronic xerostomia (Andonadou et al., 2002; Brizel et al., 2000; Vacha et al., 2003). Taken together, it appears that administration of amifostine within 30 minutes of radiotherapy or chemoradiotherapy may provide optimal benefit for cytoprotection of normal tissues.

Of primary concern with the use of any substance or technique that is intended to spare normal tissues from treatment-related toxicities is the unintended and undesirable protection of tumor cells. Clearly, procedures that protect tumors are not clinically useful. A recent meta-analysis of the available clinical data concluded that, in addition to reducing the toxicities associated with radiation therapy, amifostine does not affect the efficacy of radiotherapy (Sasse et al., 2006). To the contrary, patients receiving amifostine with radiotherapy achieved higher rates of complete response, presumably the result of fewer

Xerostomia and mucositis are significant and potentially debilitating toxicities associated with radiation therapy. The risk for these complications depends on the area receiving radiation, the dose and schedule of therapy, whether radiation therapy is combined with chemotherapy, and other factors (Sonis & Fey, 2002). Although rarely life threatening, the acute and long-term consequences can be significant, causing discomfort, reduced nutrition, and a diminished quality of life. Xerostomia is the most common toxicity associated with standard fractionated radiation therapy to the head and neck. Whereas acute xerostomia from radiation is the result of an inflammatory reaction, late xerostomia, observed 1 year after radiation, is usually a permanent result of fibrosis of the salivary gland. The dry mouth of xerostomia affects the patient's ability to eat and speak. The decreased salivary output in patients with xerostomia can be responsible for an increased risk for dental caries, oral

The results of numerous randomized controlled studies suggest that amifostine may protect against radiation- and chemoradiation-induced toxicity in patients with head and neck cancer (Table 1) (Sasse et al., 2006). In one study by Buntzel et al., 28 patients received radiation therapy in conjunction with carboplatin (Buntzel et al., 1998). Amifostine was administered to 14 patients on the day of carboplatin at a fixed dose of 500 mg (equivalent to 250–340 mg/m2). Acute grade 3 or 4 mucositis was experienced by 12 of 14 patients (86%)

patients (p < .001). Additionally, at a 12-month follow-up, 17% of patients who received amifostine experienced late grade 2 xerostomia, compared with 55% of the patients treated without amifostine (p = .05). An international phase III trial of radiation therapy with and without amifostine was conducted in 315 patients with squamous cell carcinoma of the head

treated with radiochemotherapy alone compared with none of the amifostine-treated

treatment interruptions because of reduced acute toxicity of the treatment.

**1.1 Xerostomia and oral mucositis** 

infections, and osteonecrosis.

oxidation of WR-1065 to its polyamine-like disulfide metabolite (WR-33278) is followed by a rapid consumption of oxygen in culture medium, suggesting that induction of cellular anoxia may be a mechanism for radioprotection (Purdie et al., 1983). This was supported by a study by Glover et al. that showed a rapid increase in the oxygen saturation of the venous blood after i.v. administration of amifostine without affecting the oxygen dissociation curves of hemoglobin, again suggesting that a decrease in oxygen consumption by normal tissues may be involved in amifostine-related radioprotection (Glover et al., 1984). In another study, high concentrations of WR-33278 condensed DNA, thereby limiting potential target sites for free-radical attack (Savoye et al., 1997). This activity would clearly account for a decrease in the number of double-strand breaks after radiotherapy, in turn leading to a reduction of the transient block at the G2 phase of cell division induced by radiation (Rubin et al., 1996). The enhanced cellular proliferation that results from a reduction in damage to DNA may be an important pathway to accelerated recovery of endothelial tissues that are affected soon after radiation exposure (Rubin et al., 1996) and seems to be important for the recovery of irradiated mucosa (Koukourakis et al., 1999). In addition, amifostine, indirectly through hypoxia, may upregulate the expression of a variety of proteins involved with DNA repair and inhibition of apoptosis, such as Bcl-2 and hypoxia-inducible factor-1 (Carmeliet et al., 1998; Kajstura et al., 1996; Shimizu et al., 1996).

Early phase I trials with amifostine were not able to demonstrate a maximum-tolerated dose but did establish a tolerable dose range of 740–910 mg/m2 for use in phase II studies (Blumberg et al., 1982). Amifostine is generally well tolerated, although transient adverse events may be dose related and include hypotension, nausea, vomiting, sneezing, somnolence, a metallic taste during infusion, and occasional allergic reactions that may include rash, fever, and anaphylactic shock (Blumberg et al., 1982). Although hypotension is the most clinically significant adverse event, treatment interruptions caused by a significant decline in blood pressure are rare, occurring in <5% of patients receiving amifostine. Emesis can be reduced with judicious use of an antiemetic regimen before amifostine administration. Transient hypocalcemia caused by inhibition of parathyroid hormone secretion has also been reported (Glover et al., 1983). The incidence and severity of amifostine-related adverse events have been shown to vary based on the route of administration. A recent meta-analysis of randomized studies using amifostine reported a significantly greater risk for grade 3 or 4 hypotension when amifostine was administered as a slow i.v. infusion (Sasse et al., 2006). Studies examining the s.c. administration of amifostine have demonstrated a lower incidence of hypotension and nausea/vomiting than with i.v. administration (Koukourakis et al., 2000; Anne & Curran, 2002; Anne et al., 2007). However, s.c. administration of amifostine has been reported to be associated with a higher incidence of fever and cutaneous reactions than with i.v. administration in these studies (Sasse et al., 2006; Koukourakis et al., 2000; Anne & Curran, 2002; Anne et al., 2007).

Pharmacokinetic studies in patients have demonstrated that amifostine is rapidly cleared from the plasma compartment, with a half-life of <1 minute, and >90% cleared within 6 minutes (Shaw et al., 1986). However, very little amifostine, or the metabolites WR-1065 and WR-33278, is excreted in urine 1 hour after injection. These data show that once amifostine enters the plasma, it is rapidly metabolized and distributed in the tissues, whereas the excretion of the metabolic products is very slow. Timely administration of amifostine relative to radiation or chemotherapeutic treatment is necessary. One study by Buentzel et al., in which amifostine was administered 30 minutes before chemoradiotherapy, demonstrated no significant difference in the incidence of grade 2 acute or chronic xerostomia or grade 3 oral mucositis between patients receiving i.v. amifostine and those receiving placebo (Buentzel et al., 2006). On days when combined radiochemotherapy was administered, timing between amifostine and radiotherapy may have exceeded 60 minutes. The authors suggest that timing of the amifostine doses relative to the beginning of radiotherapy may have influenced efficacy because of inadequate exposure to amifostine. In addition, the observed rates of grade 2 acute xerostomia and grade 3 oral mucositis in the placebo group were unexpectedly low, reducing the ability of the study to show significant benefit with amifostine. In contrast, studies in which amifostine was administered within 30 minutes of radiotherapy have shown promise with regard to protection from acute and chronic xerostomia (Andonadou et al., 2002; Brizel et al., 2000; Vacha et al., 2003). Taken together, it appears that administration of amifostine within 30 minutes of radiotherapy or chemoradiotherapy may provide optimal benefit for cytoprotection of normal tissues.

Of primary concern with the use of any substance or technique that is intended to spare normal tissues from treatment-related toxicities is the unintended and undesirable protection of tumor cells. Clearly, procedures that protect tumors are not clinically useful. A recent meta-analysis of the available clinical data concluded that, in addition to reducing the toxicities associated with radiation therapy, amifostine does not affect the efficacy of radiotherapy (Sasse et al., 2006). To the contrary, patients receiving amifostine with radiotherapy achieved higher rates of complete response, presumably the result of fewer treatment interruptions because of reduced acute toxicity of the treatment.

#### **1.1 Xerostomia and oral mucositis**

258 Modern Practices in Radiation Therapy

oxidation of WR-1065 to its polyamine-like disulfide metabolite (WR-33278) is followed by a rapid consumption of oxygen in culture medium, suggesting that induction of cellular anoxia may be a mechanism for radioprotection (Purdie et al., 1983). This was supported by a study by Glover et al. that showed a rapid increase in the oxygen saturation of the venous blood after i.v. administration of amifostine without affecting the oxygen dissociation curves of hemoglobin, again suggesting that a decrease in oxygen consumption by normal tissues may be involved in amifostine-related radioprotection (Glover et al., 1984). In another study, high concentrations of WR-33278 condensed DNA, thereby limiting potential target sites for free-radical attack (Savoye et al., 1997). This activity would clearly account for a decrease in the number of double-strand breaks after radiotherapy, in turn leading to a reduction of the transient block at the G2 phase of cell division induced by radiation (Rubin et al., 1996). The enhanced cellular proliferation that results from a reduction in damage to DNA may be an important pathway to accelerated recovery of endothelial tissues that are affected soon after radiation exposure (Rubin et al., 1996) and seems to be important for the recovery of irradiated mucosa (Koukourakis et al., 1999). In addition, amifostine, indirectly through hypoxia, may upregulate the expression of a variety of proteins involved with DNA repair and inhibition of apoptosis, such as Bcl-2 and hypoxia-inducible factor-1 (Carmeliet et al.,

Early phase I trials with amifostine were not able to demonstrate a maximum-tolerated dose but did establish a tolerable dose range of 740–910 mg/m2 for use in phase II studies (Blumberg et al., 1982). Amifostine is generally well tolerated, although transient adverse events may be dose related and include hypotension, nausea, vomiting, sneezing, somnolence, a metallic taste during infusion, and occasional allergic reactions that may include rash, fever, and anaphylactic shock (Blumberg et al., 1982). Although hypotension is the most clinically significant adverse event, treatment interruptions caused by a significant decline in blood pressure are rare, occurring in <5% of patients receiving amifostine. Emesis can be reduced with judicious use of an antiemetic regimen before amifostine administration. Transient hypocalcemia caused by inhibition of parathyroid hormone secretion has also been reported (Glover et al., 1983). The incidence and severity of amifostine-related adverse events have been shown to vary based on the route of administration. A recent meta-analysis of randomized studies using amifostine reported a significantly greater risk for grade 3 or 4 hypotension when amifostine was administered as a slow i.v. infusion (Sasse et al., 2006). Studies examining the s.c. administration of amifostine have demonstrated a lower incidence of hypotension and nausea/vomiting than with i.v. administration (Koukourakis et al., 2000; Anne & Curran, 2002; Anne et al., 2007). However, s.c. administration of amifostine has been reported to be associated with a higher incidence of fever and cutaneous reactions than with i.v. administration in these studies

(Sasse et al., 2006; Koukourakis et al., 2000; Anne & Curran, 2002; Anne et al., 2007).

Pharmacokinetic studies in patients have demonstrated that amifostine is rapidly cleared from the plasma compartment, with a half-life of <1 minute, and >90% cleared within 6 minutes (Shaw et al., 1986). However, very little amifostine, or the metabolites WR-1065 and WR-33278, is excreted in urine 1 hour after injection. These data show that once amifostine enters the plasma, it is rapidly metabolized and distributed in the tissues, whereas the excretion of the metabolic products is very slow. Timely administration of amifostine relative to radiation or chemotherapeutic treatment is necessary. One study by Buentzel et al., in which amifostine was administered 30 minutes before chemoradiotherapy, demonstrated no significant difference in the incidence of grade 2 acute or chronic

1998; Kajstura et al., 1996; Shimizu et al., 1996).

Xerostomia and mucositis are significant and potentially debilitating toxicities associated with radiation therapy. The risk for these complications depends on the area receiving radiation, the dose and schedule of therapy, whether radiation therapy is combined with chemotherapy, and other factors (Sonis & Fey, 2002). Although rarely life threatening, the acute and long-term consequences can be significant, causing discomfort, reduced nutrition, and a diminished quality of life. Xerostomia is the most common toxicity associated with standard fractionated radiation therapy to the head and neck. Whereas acute xerostomia from radiation is the result of an inflammatory reaction, late xerostomia, observed 1 year after radiation, is usually a permanent result of fibrosis of the salivary gland. The dry mouth of xerostomia affects the patient's ability to eat and speak. The decreased salivary output in patients with xerostomia can be responsible for an increased risk for dental caries, oral infections, and osteonecrosis.

The results of numerous randomized controlled studies suggest that amifostine may protect against radiation- and chemoradiation-induced toxicity in patients with head and neck cancer (Table 1) (Sasse et al., 2006). In one study by Buntzel et al., 28 patients received radiation therapy in conjunction with carboplatin (Buntzel et al., 1998). Amifostine was administered to 14 patients on the day of carboplatin at a fixed dose of 500 mg (equivalent to 250–340 mg/m2). Acute grade 3 or 4 mucositis was experienced by 12 of 14 patients (86%) treated with radiochemotherapy alone compared with none of the amifostine-treated

patients (p < .001). Additionally, at a 12-month follow-up, 17% of patients who received amifostine experienced late grade 2 xerostomia, compared with 55% of the patients treated without amifostine (p = .05). An international phase III trial of radiation therapy with and without amifostine was conducted in 315 patients with squamous cell carcinoma of the head

The Cytoprotective Effect of Amifostine Against Radiation Induced Toxicity 261

i.v. amifostine. 250 mg, versus RT + carboplatin (control)

i.v, amifostine, 200- 300 mg/ m2, versus RT + carboplatin (control)

Table 1. Clinical trials of amifostine therapy during radiation therapy or radiochemotherapy

and neck in which at least 75% of each parotid gland was present in the radiation fields (Brizel et al., 2000). The amifostine dose was 200 mg/m2 daily, 15–30 minutes before each fraction of radiation therapy (1.8–2.0 Gy/day, 5 days per week for 5–7 weeks, to a total dose of 50–70 Gy). Amifostine significantly reduced acute and late xerostomia and associated symptoms. Using Radiation Therapy Oncology Group (RTOG) grading criteria, patients receiving amifostine had a lower incidence of grade 2 or higher acute xerostomia (51% versus 78%; p < .001) and a lower incidence of grade 2 or higher late xerostomia (34% versus 57%; p = .002). The proportion of patients with meaningful saliva production after 1 year was significantly higher with amifostine (72% versus 49%; p = .003). Despite a trend toward lower severity of mucositis with amifostine (p = .14), the difference in the incidence of grade 3 or higher mucositis was not statistically significant (p = .48). Importantly, at 1 year, with a median follow-up of 20 months, the locoregional tumor control rates did not differ, and disease-free and overall survival times were comparable. Two-year follow-up data from this study demonstrate the continued benefits of amifostine treatment on the incidence of grade 2 xerostomia (p = .002 versus patients who did not receive amifostine) (Wasserman et al., 2005). Furthermore, no significant differences in locoregional tumor control rate, progression-free survival time, or overall survival rates were observed 2 years post-treatment between the amifostine group and the

In another study, 50 patients with head and neck cancer were randomized to receive radiotherapy plus carboplatin with or without amifostine (Antonadou et al., 2002). Treatment interruptions were more frequent in the control group. Consequently, patients receiving amifostine experienced significantly shorter treatment durations (p = .013). Patients treated with amifostine experienced less severe acute mucositis and dysphagia; all patients who did not receive amifostine in the control group experienced grade 2 mucositis by week 3. In contrast, only 9% of patients treated with amifostine experienced grade 2 mucositis (p < .001). By the fifth week, grade 4 mucositis was experienced by 52% and 4.5%

Treatment Main conclusions

i.v. amifostine treatment led to significant reduction in xerostomia compared with control treatment: reduction in mucositis was not significant between treatment groups

No difference between i.v, amifostine treatment and control treatment with regard to incidence of grade ≥2 xerostomia or grade ≥3 mucositis: low incidence of grade ≥2 xerostomia and grade ≥3 mucositis in control patients; no evidence of tumor protection was Observed with either treatment

Study Number

for head and neck cancer

control group (Wasserman et al., 2005).

of patients

Vacha et al., 2003 52 RT + carboplatin +

Bucntzel et al., 2006 132 RT + carboplatin +

Abbreviations: CRT, radiochemotherapy: RT, radiotherapy.


100 mg/m2

RT alone

alone

[26]

500 mg

RT alone

amifostine. 200 mg/m2

150 mg/m2, versus

500 mg, versus RT

200 mg/m2, versus

Brizel etal. (2000)

i.v. amifostine. 500 mg, versus RT + carboplatin (control)

i.v. amifostine, 500 mg, versus RT + carboplatin (control)

i.v. amifostine, 300 mg/m2, versus RT + carboplatin (control)

Treatment Main conclusions

Flow rates of unstimulated whole saliva recovered to 20% of baseline at

12 months post-treatment

treatment alone

i.v. amifostine treatment led to significant reduction in oral

i.v. amifostine treatment led to significant reduction in duration of acute mucositis and duration of feeding tube use compared with RT

s.c. amifostine led to significant reduction in severity of oral mucositis compared with RT treatment alone

i.v. amifostine led to significant reduction in acute and chronic xerostomia versus RT alone and increased saliva production versus RT alone; no significant reduction in grade ≥3 mucositis versus RT

i.v. amifostine led to significant decrease in severity and duration of xerostomia at 2 yrs post-treatment without compromising tumor control

Incidence of acute grade ≥2 xerostomia, 56%; 1-yr rates of locoregional tumor control,

survival, 78%, 75%, and 85%,

i.v. amifostine treatment led to significant reductions in acute xerostomia, grade ≥3 mucositis, and grade ≥3 thrombocytopenia compared

i.v. amifostine treatment had no significant effect on xerostomia or mucositis compared with control

i.v. amifostine treatment led to significant reduction in acute and late grade ≥2 xerostomia and grade ≥3 mucositis compared with control

with control treatment

respectively

treatment

treatment

progression-free survival, and overall

symptoms and duration of mucositis

Study Number

RT

RCT

of patients

McDonald et at., 1994 9 RT + i.v, amifostine,

Bourhis et al., 2000 26 RT + i.v. amifostine,

Koukourakis et al., 2000 40 RT + s.c. amifostine,

Brizel et al., 2000 315 RT + i.v. amifostine,

Wasserman et al., 2005 315 2-yr follow-up of

Anne el al., 2002, 2007 54 RT + s.c. amifostine,

Buntzel et al., 1998 39 RT + carboplatin +

Peters et al., 1999 28 RT + carboplatin +

Atttonadou et al., 2002 50 RT + carboplatin +

Wagner etal., 1998 14 RT 4· i.v.


Table 1. Clinical trials of amifostine therapy during radiation therapy or radiochemotherapy for head and neck cancer

and neck in which at least 75% of each parotid gland was present in the radiation fields (Brizel et al., 2000). The amifostine dose was 200 mg/m2 daily, 15–30 minutes before each fraction of radiation therapy (1.8–2.0 Gy/day, 5 days per week for 5–7 weeks, to a total dose of 50–70 Gy). Amifostine significantly reduced acute and late xerostomia and associated symptoms. Using Radiation Therapy Oncology Group (RTOG) grading criteria, patients receiving amifostine had a lower incidence of grade 2 or higher acute xerostomia (51% versus 78%; p < .001) and a lower incidence of grade 2 or higher late xerostomia (34% versus 57%; p = .002). The proportion of patients with meaningful saliva production after 1 year was significantly higher with amifostine (72% versus 49%; p = .003). Despite a trend toward lower severity of mucositis with amifostine (p = .14), the difference in the incidence of grade 3 or higher mucositis was not statistically significant (p = .48). Importantly, at 1 year, with a median follow-up of 20 months, the locoregional tumor control rates did not differ, and disease-free and overall survival times were comparable. Two-year follow-up data from this study demonstrate the continued benefits of amifostine treatment on the incidence of grade 2 xerostomia (p = .002 versus patients who did not receive amifostine) (Wasserman et al., 2005). Furthermore, no significant differences in locoregional tumor control rate, progression-free survival time, or overall survival rates were observed 2 years post-treatment between the amifostine group and the control group (Wasserman et al., 2005).

In another study, 50 patients with head and neck cancer were randomized to receive radiotherapy plus carboplatin with or without amifostine (Antonadou et al., 2002). Treatment interruptions were more frequent in the control group. Consequently, patients receiving amifostine experienced significantly shorter treatment durations (p = .013). Patients treated with amifostine experienced less severe acute mucositis and dysphagia; all patients who did not receive amifostine in the control group experienced grade 2 mucositis by week 3. In contrast, only 9% of patients treated with amifostine experienced grade 2 mucositis (p < .001). By the fifth week, grade 4 mucositis was experienced by 52% and 4.5%

The Cytoprotective Effect of Amifostine Against Radiation Induced Toxicity 263

undergoing radiotherapy for lung cancer [6]. However, results from the largest multicenter study conducted to date were unable to show a reduction in the incidence of esophagitis with amifostine treatment (Movsas et al., 2005). A phase III study conducted by the RTOG (trial 9801) treated 243 patients with favorable-prognosis inoperable stage II–IIIA/B non-small cell lung cancer with concurrent hyperfractionated radiotherapy plus paclitaxel (50 mg/m2) and carboplatin (dosed to achieve area under the concentration–time curve of 2) (Movsas et al., 2005). Half of the patients also received i.v. amifostine (500 mg) before the afternoon radiation treatment. During the course of the study, esophagitis was measured via National Cancer Institute Common Toxicity Criteria maximum esophagitis grade, physician dysphagia log, and patient daily self-assessment of swallowing ability. No significant differences in esophagitis were observed for patients receiving amifostine compared with those who did not receive amifostine, with the exception of the patient-reported lower rate of swallowing dysfunction observed in amifostine-treated patients (z test, p = .025). The authors attributed the lack of significant reduction in esophagitis with amifostine to several factors, including the timing of amifostine administration (Movsas et al., 2005), given that preclinical studies suggest that a single morning dose of amifostine provides superior radioprotection than with a single afternoon dose (Bachy et al., 2003; Fanzenbaker et al., 2003). The randomized trials involved in

Lower gastrointestinal mucositis frequently results from pelvic irradiation. Several clinical trials have demonstrated that amifostine pretreatment before radiotherapy or chemoradiotherapy can reduce the incidence and severity of gastrointestinal toxicities that commonly occur following these treatments (Table 2) (Koukourakis et al., 2000; Antonadou et al., 2004; Athanasiou et al., 2003; Ben-Josef et al., 2002; Dunst et al., 2000; Kouloulias et al., 2004; Kouloulias et al., 2005; Kouvaris et al., 2003; Liu et al., 1992; Simone et al., 2005; Singh et al., 2006). Guidelines published by the Mucositis Study Group of the Multinational Association of Supportive Care in Cancer/International Society for Oral Oncology recommend the use of amifostine (340 mg/m2) to prevent proctitis in patients receiving standard-dose radiotherapy (Bensadoun et al., 2006). Furthermore, these studies demonstrate that various routes of administration of amifostine (i.v., s.c., and intrarectal) are effective at reducing radiation- and chemoradiation-induced gastrointestinal toxicities in patients with pelvic malignancies. One study, conducted in 53 patients with prostate or gynecologic cancer, directly compared intrarectal amifostine administration with s.c. administration and found that intrarectal administration was more effective at reducing radiotherapy-induced rectal toxicities, whereas s.c. administration was more effective at reducing radiotherapy-induced urinary toxicities (Table 3) (Kouloulias et al., 2005). These results suggest that optimal cytoprotection may be achieved by combining routes of

Protection by amifostine against radiation-induced dermatitis was assessed in a retrospective analysis in which 100 patients with pelvic tumors treated with radiotherapy and amifostine were compared with 120 historical controls who was not administered

cytoprotection for lung irradiation are shown in table 2.

**1.3 Lower gastrointestinal mucositis** 

amifostine administration during treatment.

**1.4 Dermatitis** 

of the patients in the respective groups (p < .001). Dysphagia was similarly reduced among patients given amifostine. After 3 months of follow-up, grade 2 xerostomia was reported in 27% and 74% of patients treated with and without amifostine, respectively (p < .001).

Another consideration in the treatment of head and neck cancer is the tolerance dose of the parotid glands and the potential for raising this threshold with amifostine. Eisbruch et al. [31] reported a threshold of 26 Gy, using conformal or intensity-modulated radiotherapy, as a mean dose to spare parotid gland function (Eisbruch et al., 1999). With the use of amifostine, the threshold radiation dose for chronic xerostomia may be increased, allowing for greater dose coverage (Munter et al., 2007).

#### **1.2 Esophagitis and pneumonitis**

Damage from radiation treatment is also a major complication in the treatment of thoracic cancers, with higher rates of acute and late toxicity associated with concurrent chemoradiotherapy. Several studies have investigated the cytoprotective efficacy of amifostine against radiation-induced esophagitis and pneumonitis. Antonadou et al., in a multicenter trial of patients with advanced lung cancer, investigated whether daily pretreatment with amifostine could reduce the incidence of acute and late lung toxicity and esophagitis without affecting antitumor efficacy of radiation treatment (Antonadou et al., 2001). One hundred forty-six patients received radiotherapy in daily fractions of 2 Gy, 5 days per week, to a total of 55–60 Gy with or without daily amifostine, 340 mg/m2 15 minutes before irradiation. There was a significantly lower incidence of grade 2 or higher pneumonitis among patients receiving amifostine (9% versus 43%; p < .001). At 6 months post-treatment, fibrosis was present in 53% of patients not receiving amifostine compared with 28% of patients receiving amifostine (p < .05). The incidence of grade 2 or higher esophagitis during the fourth week of treatment was 4% among patients receiving amifostine, compared with 42% of patients receiving radiotherapy alone (p < .001). No evidence of tumor protection by amifostine was noted: complete or partial responses were observed in 75% and 76% of patients receiving amifostine or radiotherapy alone, respectively.

Komaki et al. evaluated the cytoprotective role of amifostine for esophagitis and hematologic and pulmonary toxicities in a randomized study of patients with stage II or III non-small cell lung cancer receiving concurrent chemoradiotherapy. Patients in the study group received amifostine, 500 mg i.v., twice weekly before chemoradiation, and patients in the control group received chemoradiation without Amifostine (Komaki et al., 2004). The median survival time was longer, but not significantly so, for patients receiving amifostine (26 months versus 15 months). Significantly fewer patients who received amifostine also received morphine to relieve severe esophagitis (7.4%) than patients who received chemoradiotherapy alone (31%; p = .03). Amifostine treatment was also associated with a significantly lower incidence of acute pneumonitis (3.7% versus 23%; p = .037). Although not statistically significant, 26% of patients receiving amifostine had a complete response, compared with 8% of patients who did not receive amifostine (p = .07).

Despite a limited number of studies, a recent meta-analysis reported that amifostine treatment was observed to reduce the incidence of pneumonitis and esophagitis for patients undergoing radiotherapy for lung cancer [6]. However, results from the largest multicenter study conducted to date were unable to show a reduction in the incidence of esophagitis with amifostine treatment (Movsas et al., 2005). A phase III study conducted by the RTOG (trial 9801) treated 243 patients with favorable-prognosis inoperable stage II–IIIA/B non-small cell lung cancer with concurrent hyperfractionated radiotherapy plus paclitaxel (50 mg/m2) and carboplatin (dosed to achieve area under the concentration–time curve of 2) (Movsas et al., 2005). Half of the patients also received i.v. amifostine (500 mg) before the afternoon radiation treatment. During the course of the study, esophagitis was measured via National Cancer Institute Common Toxicity Criteria maximum esophagitis grade, physician dysphagia log, and patient daily self-assessment of swallowing ability. No significant differences in esophagitis were observed for patients receiving amifostine compared with those who did not receive amifostine, with the exception of the patient-reported lower rate of swallowing dysfunction observed in amifostine-treated patients (z test, p = .025). The authors attributed the lack of significant reduction in esophagitis with amifostine to several factors, including the timing of amifostine administration (Movsas et al., 2005), given that preclinical studies suggest that a single morning dose of amifostine provides superior radioprotection than with a single afternoon dose (Bachy et al., 2003; Fanzenbaker et al., 2003). The randomized trials involved in cytoprotection for lung irradiation are shown in table 2.

#### **1.3 Lower gastrointestinal mucositis**

262 Modern Practices in Radiation Therapy

of the patients in the respective groups (p < .001). Dysphagia was similarly reduced among patients given amifostine. After 3 months of follow-up, grade 2 xerostomia was reported in

Another consideration in the treatment of head and neck cancer is the tolerance dose of the parotid glands and the potential for raising this threshold with amifostine. Eisbruch et al. [31] reported a threshold of 26 Gy, using conformal or intensity-modulated radiotherapy, as a mean dose to spare parotid gland function (Eisbruch et al., 1999). With the use of amifostine, the threshold radiation dose for chronic xerostomia may be increased, allowing

Damage from radiation treatment is also a major complication in the treatment of thoracic cancers, with higher rates of acute and late toxicity associated with concurrent chemoradiotherapy. Several studies have investigated the cytoprotective efficacy of amifostine against radiation-induced esophagitis and pneumonitis. Antonadou et al., in a multicenter trial of patients with advanced lung cancer, investigated whether daily pretreatment with amifostine could reduce the incidence of acute and late lung toxicity and esophagitis without affecting antitumor efficacy of radiation treatment (Antonadou et al., 2001). One hundred forty-six patients received radiotherapy in daily fractions of 2 Gy, 5 days per week, to a total of 55–60 Gy with or without daily amifostine, 340 mg/m2 15 minutes before irradiation. There was a significantly lower incidence of grade 2 or higher pneumonitis among patients receiving amifostine (9% versus 43%; p < .001). At 6 months post-treatment, fibrosis was present in 53% of patients not receiving amifostine compared with 28% of patients receiving amifostine (p < .05). The incidence of grade 2 or higher esophagitis during the fourth week of treatment was 4% among patients receiving amifostine, compared with 42% of patients receiving radiotherapy alone (p < .001). No evidence of tumor protection by amifostine was noted: complete or partial responses were observed in 75% and 76% of patients receiving amifostine or radiotherapy alone,

Komaki et al. evaluated the cytoprotective role of amifostine for esophagitis and hematologic and pulmonary toxicities in a randomized study of patients with stage II or III non-small cell lung cancer receiving concurrent chemoradiotherapy. Patients in the study group received amifostine, 500 mg i.v., twice weekly before chemoradiation, and patients in the control group received chemoradiation without Amifostine (Komaki et al., 2004). The median survival time was longer, but not significantly so, for patients receiving amifostine (26 months versus 15 months). Significantly fewer patients who received amifostine also received morphine to relieve severe esophagitis (7.4%) than patients who received chemoradiotherapy alone (31%; p = .03). Amifostine treatment was also associated with a significantly lower incidence of acute pneumonitis (3.7% versus 23%; p = .037). Although not statistically significant, 26% of patients receiving amifostine had a complete response,

Despite a limited number of studies, a recent meta-analysis reported that amifostine treatment was observed to reduce the incidence of pneumonitis and esophagitis for patients

compared with 8% of patients who did not receive amifostine (p = .07).

27% and 74% of patients treated with and without amifostine, respectively (p < .001).

for greater dose coverage (Munter et al., 2007).

**1.2 Esophagitis and pneumonitis** 

respectively.

Lower gastrointestinal mucositis frequently results from pelvic irradiation. Several clinical trials have demonstrated that amifostine pretreatment before radiotherapy or chemoradiotherapy can reduce the incidence and severity of gastrointestinal toxicities that commonly occur following these treatments (Table 2) (Koukourakis et al., 2000; Antonadou et al., 2004; Athanasiou et al., 2003; Ben-Josef et al., 2002; Dunst et al., 2000; Kouloulias et al., 2004; Kouloulias et al., 2005; Kouvaris et al., 2003; Liu et al., 1992; Simone et al., 2005; Singh et al., 2006). Guidelines published by the Mucositis Study Group of the Multinational Association of Supportive Care in Cancer/International Society for Oral Oncology recommend the use of amifostine (340 mg/m2) to prevent proctitis in patients receiving standard-dose radiotherapy (Bensadoun et al., 2006). Furthermore, these studies demonstrate that various routes of administration of amifostine (i.v., s.c., and intrarectal) are effective at reducing radiation- and chemoradiation-induced gastrointestinal toxicities in patients with pelvic malignancies. One study, conducted in 53 patients with prostate or gynecologic cancer, directly compared intrarectal amifostine administration with s.c. administration and found that intrarectal administration was more effective at reducing radiotherapy-induced rectal toxicities, whereas s.c. administration was more effective at reducing radiotherapy-induced urinary toxicities (Table 3) (Kouloulias et al., 2005). These results suggest that optimal cytoprotection may be achieved by combining routes of amifostine administration during treatment.

#### **1.4 Dermatitis**

Protection by amifostine against radiation-induced dermatitis was assessed in a retrospective analysis in which 100 patients with pelvic tumors treated with radiotherapy and amifostine were compared with 120 historical controls who was not administered

The Cytoprotective Effect of Amifostine Against Radiation Induced Toxicity 265

**P Value**  **Remarks** 

0.044 Nonrandomized (intravenous)

<0.001 Nonrandomized

0.0325 Nonrandomized (intrarectal)

<0.05 Randomized

<0.05 Nonrandomized

<0.011 Randomized

0.03 Randomized (intravenous)

<0.01 Randomized (intravenous)

<0.001 Randomized (intravenous)

0.001 Randomized (intravenous)

0.026 Randomized (intrarectal)

0.04 Randomized (subcutaneous vs intrarectal)

(retrospective, intravenous)

**Author N Rectal Toxicity (Control vs** 

Liu et al., 1992 100 14% vs 0% ; moderate or

Dunst et al., 2000

Antonadou et al., 2004

Kligerman et al.,

Kouvaris et al.,

Ben-Josef et al.,

Koukourakis et al., 2000

Kouvaris et al.,

Muller et al., 2004

Athanasiou et al., 2003

Kouloulias et al., 2004

Singh et al., 2006; Simone et al., 2005

Kouloulias et al., 2005

**2.1 i.v. Amifostine** 

IR=intrarectal; s.c.=subcutneous

**2. Different ways of administration** 

1992

2002

2002

2003

**Amifostine)** 

30 1.07±1.03 vs 0.40±0.63;

for control

late toxicity

42%

11%

severe late toxicities

124 5.6% for amifostine vs 22.2%

220 Grade I/II toxicity, 70% vs

15% (1500–2500 mg amifostine)

40 Grade III/IV observed in 15% for control vs 0% for

amifosine group

measurements)

radiation)

205 Grade II/III acute toxicity, 22.1% vs 5.5% (3rd wk of

67 Grade I/II acute toxicity, 44% vs 15% for IR

53 Grade I/II acute toxicity, 42% for s.c. Vs 11% for IR

Table 3. Clinical Trials of Amifostine With Radiotherapy in Pelvic Tumors

measured before and immediately after the 3-minute amifostine infusion.

36 Grade I/II toxicity, 88% vs

6 Leukocytes and lymphocytes irradiated were

radioprotected (comet assay

30 33% in 1gr IR vs 0% in 2gr IR 0.06 Randomized

The American Society of Clinical Oncology guidelines for cytoprotective agents recommend amifostine at a dose of 200 mg/m2 daily, given as a slow i.v. push over 3 minutes, 15–30 minutes before each fraction of radiation therapy (Hensley et al., 1999). Adverse events are reduced at this lower dose. Nonetheless, administration of amifostine requires close patient monitoring. Many patients require antiemetics. Hypotension associated with amifostine at this dose is less frequent but still requires close monitoring. Blood pressure should be

29 Grade I/II toxicity, 50%

100 5% vs 0% moderate or severe

(500–1000 mg amifostine) vs

maximum diarrhea score


*Abbreviations:* P = paclitaxel; C = carboplatin; RT = radiotherapy; NCI-CTC = National Cancer Institute-Common Toxicity Criteria.

Table 2. Randomized trials with amifostine in lung cancer

amifostine (Kouvaris et al., 2002). There was a 77% lower risk for radiation-induced dermatitis with amifostine use. The severity of dermatitis was also significantly lower among patients receiving amifostine compared with historical controls: the mean gross dermatitis scores were 0.18 ± 0.09 versus 1.0 ± 0.11 (p < .001). In another study of 40 patients receiving radiation treatment for pelvic tumors, grade 2 or 3 dermatitis of the perineal/vulvar area was observed in all patients with gynecologic and rectal cancer who did not receive amifostine (500 mg s.c.) (Koukourakis et al., 20000). Among patients who received amifostine, only grade 1 dermatitis was noted.



IR=intrarectal; s.c.=subcutneous

Table 3. Clinical Trials of Amifostine With Radiotherapy in Pelvic Tumors

#### **2. Different ways of administration**

#### **2.1 i.v. Amifostine**

264 Modern Practices in Radiation Therapy

Induction PC 500 mg i.v.

4doses/wk between RT fractions

each chemo (Days 1, 22, 43, 50, 57,64,

500 mg i.v. before weekly chemo; 200 mg i.v. daily before RT

71, 78)

before RT

300 mg/m2/d before

chemoradiation and RT

500 mg i.v. 1st, 2nd day each wk before chemo and 1st RT fraction

No difference by NCI-CTC esophagitis, Swallowing diaries (*p* < 0.03) and weight loss (*p*  < 0.05) favour amifostine (median survival, 15.6

Esophagitis Grade 2–3: 43% in amifostine, 70% in control (not significant) (median survival, 12.5 and 14.5

No difference in toxicity, no survival data (ongoing trial)

↓esophagitis (*p* <0.001) ↓pneumonitis (*p* = 0.009) (no survival data)

↓Degree of esophagitis, ↓Pneumonitis, ↓Neutropenic fever (median survival,19 and

and 15.8 mo)

↓Pneumonitis ↓Esophagitis (no survival data)

20 mo)

mo)

Reference Radiation dose Chemotherapy Amifostine dose Comment

60–66 Gy at 2.0 Gy Induction PC 740 mg/m2 with

gemcitabine and cisplatin X 3 after chemoradiation

55–60 Gy at 2.0 Gy. None 340 mg/m2/d

weekly Por C

Concurrent i.v. cisplatin Days 1, 8,29, 36; Oral etoposide Days1–5 8–12, 29–33,36–40

*Abbreviations:* P = paclitaxel; C = carboplatin; RT = radiotherapy; NCI-CTC = National Cancer Institute-

amifostine (Kouvaris et al., 2002). There was a 77% lower risk for radiation-induced dermatitis with amifostine use. The severity of dermatitis was also significantly lower among patients receiving amifostine compared with historical controls: the mean gross dermatitis scores were 0.18 ± 0.09 versus 1.0 ± 0.11 (p < .001). In another study of 40 patients receiving radiation treatment for pelvic tumors, grade 2 or 3 dermatitis of the perineal/vulvar area was observed in all patients with gynecologic and rectal cancer who did not receive amifostine (500 mg s.c.) (Koukourakis et al., 20000). Among patients who

64.8 Gy at 1.8 Gy Concurrent PC,

55–60 Gy at 2.0Gy. Concurrent

Table 2. Randomized trials with amifostine in lung cancer

received amifostine, only grade 1 dermatitis was noted.

69.6 Gy at 1.2 Gy (hyperfractionation)

Movsas et al.,

Leong et al. , 2003 (*n* = 60)

Senzer et al., 2002 (*n* = 63)

Antonadou et al., 2001 (*n* = 146)

Antonadou et al., 2003 (*n* = 73)

Komaki et al.,

Common Toxicity Criteria.

2004 (*n* = 62) 69.6 Gy at 1.2 Gy (hyperfractionation)

2005 (*n* = 242)

> The American Society of Clinical Oncology guidelines for cytoprotective agents recommend amifostine at a dose of 200 mg/m2 daily, given as a slow i.v. push over 3 minutes, 15–30 minutes before each fraction of radiation therapy (Hensley et al., 1999). Adverse events are reduced at this lower dose. Nonetheless, administration of amifostine requires close patient monitoring. Many patients require antiemetics. Hypotension associated with amifostine at this dose is less frequent but still requires close monitoring. Blood pressure should be measured before and immediately after the 3-minute amifostine infusion.

The Cytoprotective Effect of Amifostine Against Radiation Induced Toxicity 267

in treatment for radiation-induced toxicities were required in that study. A combination of intrarectal and s.c. amifostine administration might be optimal for cytoprotection with pelvic

The past years all the trials with amifostine and pelvic radiotherapy used as endpoints the

Increase of 4– 6 stools per d, or nocturnal stools, or moderate cramping

Diarrhea requiring parasympatholytic drugs, mucous discharge not necessitating sanitary pads, rectal or abdominal pain requiring analgesics

EORTC-RTOG= European Organization for Research and Treatment of Cancer/Radiation Therapy

Table 4. WHO Toxicity Criteria and RTOG Acute Radiation Morbidity Scoring Criteria

A specific analytical for subjective and objective measurements was introduced. Endoscopy offers accurate endpoints for the evaluation of tissue damage, whereas the criteria of rectosigmoidoscopy findings are still not well defined in the literature. The literature deals mainly with symptomatic patients presenting with rectal bleeding, pain, increased stool frequency, urgency and incontinence, whereas systematic endoscopic analysis including asymptomatic patients rarely exists. A valid scoring system is essential for adequate description of acute rectal toxicity. For the benefit of sharing and comparing data collected from endoscopy after RT, we have introduced a graduation system based on

**Grade 1 Grade 2 Grade 3 Grade 4** 

Increase of 7–9 stools per d, or incontinence, or severe cramping

Diarrhea requiring parenteral support, severe mucous or blood discharge necessitating sanitary pads/abdomin al distension (flat plate radiograph demonstrates distended bowel loops)

Increase of >10 stools per d or grossly bloody diarrhea, or need for parenteral

Acute or subacute obstruction, fistula or

support

perforation; gastrointestinal bleeding requiring transfusion; abdominal pain or tenesmus requiring tube decompression or bowel diversion

**3. New grading scale for acute pelvic radiation induced toxicity** 

WHO and RTOG/EORTC scales as shown in table 4.

None Increase of 2–3 stools per d over

None Increased

pretreatment

frequency or change in quality of bowel habits not requiring medication, rectal

discomfort not requiring analgesics

Oncology Group; WHO=World Health Organization.

irradiation.

WHO Toxicity Grade

EORTC-RTOG scale for lower gastrointestinal

 **Grade** 

**0** 

#### **2.2 s.c. Amifostine**

The s.c. administration of amifostine has been proposed to reduce treatment-related and dose-limiting adverse events (Koukourakis et al., 20000). In a pharmacokinetic study, the plasma concentration of WR-1605 after s.c. injection of 500 mg of amifostine was 67% of that after a 200 mg/m2 i.v. dose (Shaw et al., 1997). Lower plasma levels of amifostine after s.c. injection do not necessarily translate to lower tissue concentrations. Because the amount of amifostine that is absorbed and converted to the active metabolites is not dependent on plasma pharmacokinetics, i.v. or s.c. administration may not have a significant impact on whether therapeutic levels are achieved in the tissues. Precise determination of the protective efficacy of different routes of administration will require more comprehensive studies that measure intracellular levels of the metabolites or assess radiation-induced DNA double-strand breaks in tissues after i.v. or s.c. administration of amifostine. Nonetheless, the efficacy of s.c. amifostine administration is best addressed in the context of a clinical trial.

A phase II randomized trial with 140 patients assessed the feasibility, tolerance, and activity of the s.c. route (Koukourakis et al., 20000). A dose of amifostine of 500 mg s.c. was administered 20 minutes before each fraction of radiotherapy. The s.c. administration of amifostine was well tolerated by 85% of patients. In approximately 15% of patients, amifostine therapy was interrupted because of cumulative asthenia or a fever/rash reaction. Mild nausea was frequent (29%), and the incidence of hypotension was negligible (3%). Significantly less pharyngeal, esophageal, and rectal mucositis was observed among patients receiving amifostine (*p* < .04). Treatment delays because of grade 3 mucositis were significantly longerin patients treated with radiotherapy alone (*p* < .04).

#### **2.3 Endorectal**

Initial attempts with rectal administration of amifostine admixed in a foam did not demonstrate protection in patients receiving large pelvic fields of radiation (Montana et al., 1992). However, after successful topical application of amifostine in the rectum of male rats (Ben-Josef et al., 1995), subsequent significant clinical benefit of endorectal administration of amifostine was demonstrated in a phase I study (Ben-Josef et al., 2002).

A randomized trial of 67 patients undergoing radiotherapy for prostate cancer further assessed intrarectal administration of amifostine (Kouloulias et al., 2002). Patients were treated with or without amifostine at a dose of 1,500 mg intrarectally 20–30 minutes before each radiotherapy session. All patients receiving amifostine completed therapy without amifostine-related toxicities, suggesting that intrarectal amifostine was feasible and well tolerated. According to RTOG grading criteria, amifostine was superior to no treatment, with a significantly lower incidence of rectal mucositis (15% versus 44%; *p* < .04). The mean rectal mucositis index of patients who received amifostine was 0.3 ± 0.1 compared with 2.2 ± 0.4 in patients without cytoprotection (*p* < .001). The severity of rectal mucositis was significantly lower in patients who received amifostine (*p* < .001). Urinary toxicity was comparable between the two groups (*p* = .76). A more recent study suggests that the efficacy of intrarectal amifostine may be dose dependent. Although not statistically significant, the incidence of acute grade 2 rectal mucositis was lower in patients receiving a 2-g suspension of amifostine (*n* = 12) than in those receiving 1 g (*n* = 18; *p* = .06) (Sigh et al., 2006). No breaks

The s.c. administration of amifostine has been proposed to reduce treatment-related and dose-limiting adverse events (Koukourakis et al., 20000). In a pharmacokinetic study, the plasma concentration of WR-1605 after s.c. injection of 500 mg of amifostine was 67% of that after a 200 mg/m2 i.v. dose (Shaw et al., 1997). Lower plasma levels of amifostine after s.c. injection do not necessarily translate to lower tissue concentrations. Because the amount of amifostine that is absorbed and converted to the active metabolites is not dependent on plasma pharmacokinetics, i.v. or s.c. administration may not have a significant impact on whether therapeutic levels are achieved in the tissues. Precise determination of the protective efficacy of different routes of administration will require more comprehensive studies that measure intracellular levels of the metabolites or assess radiation-induced DNA double-strand breaks in tissues after i.v. or s.c. administration of amifostine. Nonetheless, the efficacy of s.c. amifostine administration is best addressed in the context of a clinical trial.

A phase II randomized trial with 140 patients assessed the feasibility, tolerance, and activity of the s.c. route (Koukourakis et al., 20000). A dose of amifostine of 500 mg s.c. was administered 20 minutes before each fraction of radiotherapy. The s.c. administration of amifostine was well tolerated by 85% of patients. In approximately 15% of patients, amifostine therapy was interrupted because of cumulative asthenia or a fever/rash reaction. Mild nausea was frequent (29%), and the incidence of hypotension was negligible (3%). Significantly less pharyngeal, esophageal, and rectal mucositis was observed among patients receiving amifostine (*p* < .04). Treatment delays because of grade 3 mucositis were

Initial attempts with rectal administration of amifostine admixed in a foam did not demonstrate protection in patients receiving large pelvic fields of radiation (Montana et al., 1992). However, after successful topical application of amifostine in the rectum of male rats (Ben-Josef et al., 1995), subsequent significant clinical benefit of endorectal administration of

A randomized trial of 67 patients undergoing radiotherapy for prostate cancer further assessed intrarectal administration of amifostine (Kouloulias et al., 2002). Patients were treated with or without amifostine at a dose of 1,500 mg intrarectally 20–30 minutes before each radiotherapy session. All patients receiving amifostine completed therapy without amifostine-related toxicities, suggesting that intrarectal amifostine was feasible and well tolerated. According to RTOG grading criteria, amifostine was superior to no treatment, with a significantly lower incidence of rectal mucositis (15% versus 44%; *p* < .04). The mean rectal mucositis index of patients who received amifostine was 0.3 ± 0.1 compared with 2.2 ± 0.4 in patients without cytoprotection (*p* < .001). The severity of rectal mucositis was significantly lower in patients who received amifostine (*p* < .001). Urinary toxicity was comparable between the two groups (*p* = .76). A more recent study suggests that the efficacy of intrarectal amifostine may be dose dependent. Although not statistically significant, the incidence of acute grade 2 rectal mucositis was lower in patients receiving a 2-g suspension of amifostine (*n* = 12) than in those receiving 1 g (*n* = 18; *p* = .06) (Sigh et al., 2006). No breaks

significantly longerin patients treated with radiotherapy alone (*p* < .04).

amifostine was demonstrated in a phase I study (Ben-Josef et al., 2002).

**2.2 s.c. Amifostine** 

**2.3 Endorectal** 

in treatment for radiation-induced toxicities were required in that study. A combination of intrarectal and s.c. amifostine administration might be optimal for cytoprotection with pelvic irradiation.

### **3. New grading scale for acute pelvic radiation induced toxicity**

The past years all the trials with amifostine and pelvic radiotherapy used as endpoints the WHO and RTOG/EORTC scales as shown in table 4.


EORTC-RTOG= European Organization for Research and Treatment of Cancer/Radiation Therapy Oncology Group; WHO=World Health Organization.

Table 4. WHO Toxicity Criteria and RTOG Acute Radiation Morbidity Scoring Criteria

A specific analytical for subjective and objective measurements was introduced. Endoscopy offers accurate endpoints for the evaluation of tissue damage, whereas the criteria of rectosigmoidoscopy findings are still not well defined in the literature. The literature deals mainly with symptomatic patients presenting with rectal bleeding, pain, increased stool frequency, urgency and incontinence, whereas systematic endoscopic analysis including asymptomatic patients rarely exists. A valid scoring system is essential for adequate description of acute rectal toxicity. For the benefit of sharing and comparing data collected from endoscopy after RT, we have introduced a graduation system based on

The Cytoprotective Effect of Amifostine Against Radiation Induced Toxicity 269

and the normal interval between administration of amifostine and radiotherapy may explain these differences. Nonhomogenous distribution of amifostine and its metabolites within a tissue, even at the level of the DNA (savoye et al., 1997), may also contribute to this

Fig. 1. Rectosigmoidoscopic findings. Panels A and B illustrate a regular rectal mucosa in patients after intrarectal administration of amifostine. Panels C and D are from patients who did not receive amifostine and illustrate congested mucosa with superficial ulceration >1

The U.S. Food and Drug Administration has approved the i.v. use of amifostine to reduce the cumulative renal toxicity associated with repeated administration of cisplatin in patients with advanced ovarian cancer and to reduce the incidence of moderate to severe xerostomia in patients undergoing postoperative radiation treatment for head and neck cancer, where the radiation port includes a substantial portion of the parotid glands. Amifostine has been

heterogeneity.

cm2 (indicated by the arrows).


rectosigmoidoscopic criteria focused on acute effects and standardized terminology published by the European Society for Gastrointestinal Endoscopy. The scale is shown in table 5.

\*Modification to Subjective Objective Management Analytic scale to fit radiation-induced acute toxicity to the rectum. Subjective and objective items were used for evaluation of acute radiation-induced rectal mucositis. The second and third items of the objective scale were based on findings from flexible rectosigmoidoscopy and were in accordance with the endoscopic terminology of the World Organization for Digestive Endoscopy. The final score was the sum of scores of the 8 items (score=0 in the absence of toxicity).

Table 5. Rectal Toxicity Grade\*

Our experience has shown that the terminology is practicable and provides a definition of terms usable by radiation oncologist and endoscopists. The S-RS scale showed a satisfied clinical validity and reliability (Kouvaris etal., 2003). In a previous publication (Kouloulias et al., 2004) the rectosigmoidoscopic findings for amifostine versus no amifostine showed significant differences, as shown in figure 1.

#### **4. Conclusion**

Normal tissues vary in the extent that they are protected from radiation damage by amifostine. Because amifostine does not cross the blood–brain barrier, the central nervous system, often the dose-limiting organ in radiotherapy, is not protected (Millar et al., 1982; Washburn et al., 1976). Protection factors for other tissues range from three in the hematopoietic system and salivary glands to approximately one in the lung, kidney, and bladder (Rojas et al., 1984; Rojas et al., 1986; Travis 1984). Within the same tissues, a range of protection factors has been reported (Rojas et al., 1984; Mori et al., 1984). Discrepancies in WR-1065 concentrations in tissues within 15–30 minutes of administration (Utley et al., 1984)

rectosigmoidoscopic criteria focused on acute effects and standardized terminology published by the European Society for Gastrointestinal Endoscopy. The scale is shown in

urgency

Mucosal loss Occasional Intermittent Persistent Refractory

Stool frequency 2–4 per d 4–8 per d >8 per d Uncontrolled

per wk

cm2

Punctate, congested mucosa

\*Modification to Subjective Objective Management Analytic scale to fit radiation-induced acute toxicity to the rectum. Subjective and objective items were used for evaluation of acute radiation-induced rectal mucositis. The second and third items of the objective scale were based on findings from flexible rectosigmoidoscopy and were in accordance with the endoscopic terminology of the World

Organization for Digestive Endoscopy. The final score was the sum of scores of the 8 items (score=0 in

Our experience has shown that the terminology is practicable and provides a definition of terms usable by radiation oncologist and endoscopists. The S-RS scale showed a satisfied clinical validity and reliability (Kouvaris etal., 2003). In a previous publication (Kouloulias et al., 2004) the rectosigmoidoscopic findings for amifostine versus no amifostine showed

Normal tissues vary in the extent that they are protected from radiation damage by amifostine. Because amifostine does not cross the blood–brain barrier, the central nervous system, often the dose-limiting organ in radiotherapy, is not protected (Millar et al., 1982; Washburn et al., 1976). Protection factors for other tissues range from three in the hematopoietic system and salivary glands to approximately one in the lung, kidney, and bladder (Rojas et al., 1984; Rojas et al., 1986; Travis 1984). Within the same tissues, a range of protection factors has been reported (Rojas et al., 1984; Mori et al., 1984). Discrepancies in WR-1065 concentrations in tissues within 15–30 minutes of administration (Utley et al., 1984)

Intermittent & tolerable

**Grade 1 Grade 2 Grade 3 Grade 4** 

Occasional Intermittent Persistent Refractory

Persistent urgency

Persistent & intense

Diffused, congested mucosa

Persistent, daily Gross

Deep ulcer Surgical

Refractory

diarrhoea

Refractory & excruciating

hemorrhage

intervention

Bleeding mucosa

table 5.

Subjective

Sphincter control

Objective

the absence of toxicity).

**4. Conclusion** 

Table 5. Rectal Toxicity Grade\*

significant differences, as shown in figure 1.

Pain Occasional &

Mucosa surface Localized spotted,

minimal

Bleeding Occult Occasionally >2

congested mucosa

Ulceration Superficial ≤1 cm2 Superficial >1

Tenesmus Occasional urgency Intermittent

and the normal interval between administration of amifostine and radiotherapy may explain these differences. Nonhomogenous distribution of amifostine and its metabolites within a tissue, even at the level of the DNA (savoye et al., 1997), may also contribute to this heterogeneity.

Fig. 1. Rectosigmoidoscopic findings. Panels A and B illustrate a regular rectal mucosa in patients after intrarectal administration of amifostine. Panels C and D are from patients who did not receive amifostine and illustrate congested mucosa with superficial ulceration >1 cm2 (indicated by the arrows).

The U.S. Food and Drug Administration has approved the i.v. use of amifostine to reduce the cumulative renal toxicity associated with repeated administration of cisplatin in patients with advanced ovarian cancer and to reduce the incidence of moderate to severe xerostomia in patients undergoing postoperative radiation treatment for head and neck cancer, where the radiation port includes a substantial portion of the parotid glands. Amifostine has been

The Cytoprotective Effect of Amifostine Against Radiation Induced Toxicity 271

Bardet E, Martin L, Calais G, et al. Subcutaneous compared with intravenous administration

Ben-Josef E, Han S, Tobi M et al. A pilot study of topical intrarectal application of amifostine

Ben-Josef E, Mesina J, Shaw LM et al. Topical application of WR-2721 achieves high

Bensadoun RJ, Schubert MM, Lalla RV et al. Amifostine in the management of radiationinduced and chemo-induced mucositis. Support Care Cancer 2006;14:566–572. Blumberg AL, Nelson DF, Gramkowski M et al. Clinical trials of WR-2721 with radiation

Bourhis J, Blanchard P, Maillard E, et al. Effect of Amifostine on Survival Among Patients

Bourhis J, De Crevoisier R, Abdulkarim B et al. A randomized study of very accelerated

Brizel DM, Wasserman TH, Henke M et al. Phase III randomized trial of amifostine as a radioprotector in head and neck cancer. J Clin Oncol 2000;18:3339–3345. Buentzel J, Micke O, Adamietz IA et al. Intravenous amifostine during chemoradiotherapy

Buntzel J, Glatzel M, Frohlich D et al. Intensification of radiochemotherapy with Ethyol in

Buntzel J, Kuttner K, Frohlich D et al. Selective cytoprotection with amifostine in concurrent radiochemotherapy for head and neck cancer. Ann Oncol 1998;9:505–509. Calabro-Jones PM, Fahey RC, Smoluk GD et al. Alkaline phosphatase promotes

Carmeliet P, Dor Y, Herbert JM et al. Role of HIF-1alpha in hypoxia-mediated apoptosis, cell

Dunst J, Semlin S, Pigorsch S et al. Intermittent use of amifostine during postoperative

Eisbruch A, Ten Haken RK, Kim HM et al. Dose, volume, and function relationships in

Glover D, Negendank W, Delivoria-Papadopoulos M et al. Alterations in oxygen transport following WR-2721. Int J Radiat Oncol Biol Phys 1984;10:1565–1568.

proliferation and tumour angiogenesis. Nature 1998;394:485–490.

head and neck cancer. Int J Radiat Oncol Biol Phys 1999;45:577–587. Fazenbaker CA, Bachy CM, Kifle G et al. Dose and schedule dependency of amifostine

Treated With Radiotherapy: A Meta-Analysis of Individual Patient Data. J Clin

radiotherapy with and without amifostine in head and neck squamous cell

for head-and-neck cancer: A randomized placebo-controlled phase III study. Int J

radioprotection and accumulation of WR-1065 in V79–171 cells incubated in medium containing WR-2721. Int J Radiat Biol Relat Stud Phys Chem Med

radiochemotherapy and acute toxicity in rectal cancer patients. Strahlenther Onkol

parotid salivary glands following conformal and intensity-modulated irradiation of

protection against hyperfractionated radiotherapy in a rat model. Proc Am Soc Clin

concentrations in the rectal wall. Radiat Res 1995;143:107–110.

therapy. Int J Radiat Oncol Biol Phys 1982;8:561–563.

carcinoma. Int J Radiat Oncol Biol Phys 2000;46:1105–1108.

head and neck cancer. Proc Am Soc Clin Oncol 1998;17:403A.

Radiat Oncol Biol Phys 2006;64:684–691.

10;29(2):127-33.

2002;53:1160–1164.

Oncol. 2011 May 16.

1985;47:23–27.

2000;176:416–421.

Oncol 2003;22:518.

of amifostine in patients with head and neck cancer receiving radiotherapy: final results of the GORTEC2000-02 phase III randomized trial.J Clin Oncol. 2011 Jan

for prevention of late radiation rectal injury. Int J Radiat Oncol Biol Phys

proven to be a useful addition to the arsenal of the radiation oncologist, helping improve patients' quality of life and in some cases allowing more aggressive radio- and chemotherapeutic regimens. Currently, s.c. administration of amifostine is a standard practice for patients with head and neck cancer as well as for patients with recurrent ovarian carcinoma (Hensley et al., 2009), while there is evidence now that the s.c is not superior to i.v. administration of amifostine (Bardet et al., 2011). Refinements in doses and administration of amifostine lead to constant improvement in the adverse event profile, resulting in fewer interruptions in treatment and ultimately improving patient outcomes (Jellena et al., 2006; Bourhis et al., 2011). At last but not least, recent meta-analysis from 16 randomized trials (1,554 patients) confirmed the lack of any tumor protection in routine radiotherapy practice when amifostine is administered (Bourhis et al., 2011).

It has to be mentioned that the graduation system designed by our group is user-friendly and more than this, it is an interface between radiation-oncologists and gastroenterologists by means of common terminology between specializations for radiation induced rectal toxicity.

#### **5. References**


proven to be a useful addition to the arsenal of the radiation oncologist, helping improve patients' quality of life and in some cases allowing more aggressive radio- and chemotherapeutic regimens. Currently, s.c. administration of amifostine is a standard practice for patients with head and neck cancer as well as for patients with recurrent ovarian carcinoma (Hensley et al., 2009), while there is evidence now that the s.c is not superior to i.v. administration of amifostine (Bardet et al., 2011). Refinements in doses and administration of amifostine lead to constant improvement in the adverse event profile, resulting in fewer interruptions in treatment and ultimately improving patient outcomes (Jellena et al., 2006; Bourhis et al., 2011). At last but not least, recent meta-analysis from 16 randomized trials (1,554 patients) confirmed the lack of any tumor protection in routine

It has to be mentioned that the graduation system designed by our group is user-friendly and more than this, it is an interface between radiation-oncologists and gastroenterologists by means of common terminology between specializations for radiation induced rectal

American Cancer Society. Cancer Facts and Figures 2005. Atlanta, GA: American Cancer

Anne PR, Curran WJ Jr. A phase II trial of subcutaneous amifostine and radiation therapy in patients with head and neck cancer. Semin Radiat Oncol 2002;12(suppl 1):18–19. Anne PR, Machtay M, Rosenthal DI et al. A Phase II trial of subcutaneous amifostine and

Antonadou D, Athanassiou H, Sarris N et al. Final results of a randomized phase III trial of

Antonadou D, Coliarakis N, Synodinou M et al. Randomized phase III trial of radiation

Antonadou D, Pepelassi M, Synodinou M et al. Prophylactic use of amifostine to prevent

Antonadou D, Throuvalas N, Petridis A, et al. Effect of amifostine on toxicities associated

Athanassiou H, Antonadou D, Coliarakis N et al. Protective effect of amifostine during

Bachy CM, Fazenbaker CA, Kifle G et al. Daily dosing with amifostine is necessary for full

Protection and pharmacokinetics. Proc Am Soc Clin Oncol 2003;22:518.

randomized trial. Int J Radiat Oncol Biol Phys 2003;56:1154–1160.

radiation therapy in patients with head-and-neck cancer. Int J Radiat Oncol Biol

chemoradiation treatment + amifostine in patients with colorectal cancer: Clinical Radiation Oncology Hellenic Group. Presented at the 46th Annual Meeting of the American Society for Therapeutic Radiology and Oncology; October 3–7; Atlanta,

treatment +/- amifostine in patients with advanced-stage lung cancer. Int J Radiat

radiochemotherapy-induced mucositis and xerostomia in head-and-neck cancer.

with radiochemotherapy in patients with locally advanced non-small-cell lung

fractionated radiotherapy in patients with pelvic carcinomas: Results of a

protection against oral mucositis caused by fractionated radiation in rats:

radiotherapy practice when amifostine is administered (Bourhis et al., 2011).

toxicity.

**5. References** 

Society, 2005:60.

Phys 2007;67:445–452.

Oncol Biol Phys 2001;51:915–922.

Int J Radiat Oncol Biol Phys 2002;52:739–747.

cancer. Int J Radiat Oncol Biol Phys 2003;57:402–408.

GA, 2004.


The Cytoprotective Effect of Amifostine Against Radiation Induced Toxicity 273

Liu T, Liu Y, He S et al. Use of radiation with or without WR-2721 in advanced rectal cancer.

McDonald S, Meyerowitz C, Smudzin T et al. Preliminary results of a pilot study using WR-

Millar JL, McElwain TJ, Clutterbuck RD et al. The modification of melphalan toxicity in

Montana GS, Anscher MS, Mansbach CM 2nd et al. Topical application of WR-2721 to

Mori T, Nikaido O, Sugahara T. Dephosphorylation of WR-2721 with mouse tissue

Movsas B, Scott C, Langer C et al. Randomized trial of amifostine in locally advanced non-

Müller AC, Pigorsch S, Beyer C, et al. Radioprotective effects of amifostine in vitro and in vivo measured with the comet assay. Strahlenther Onkol. 2004 Aug;180(8):517-25. Munter MW, Hoffner S, Hof H et al. Changes in salivary gland function after radiotherapy

Peters K, Mucke R, Hamann D et al. Supportive use of amifostine in patients with head and

the application of amifostine? Strahlenther Onkol 1999;175(suppl 4):23–26. Purdie JW, Inhaber ER, Schneider H et al. Interaction of cultured mammalian cells with WR-

Rojas A, Denekamp J. The influence of X ray dose levels on normal tissue radioprotection by

Rojas A, Stewart FA, Soranson JA et al. Fractionation studies with WR-2721: Normal tissues

Rubin DB, Drab EA, Kang HJ et al. WR-1065 and radioprotection of vascular endothelial cells. I. Cell proliferation, DNA synthesis and damage Radiat Res, 1996;145:210–216. Sasse AD, Clark LG, Sasse EC et al. Amifostine reduces side effects and improves complete

Savoye C, Swenberg C, Hugot S et al. Thiol WR-1065 and disulphide WR-33278, two

Senzer N. A phase III randomized evaluation of amifostine in stage IIIA/IIIB non-small cell

Radiat Biol Relat Stud Phys Chem Med 1983;43:517–527.

WR-2721. Int J Radiat Oncol Biol Phys 1984;10:2351–2356.

induced strand breakage. Int J Radiat Biol 1997;71:193–202.

and tumour. Radiother Oncol 1986;6:51–60.

Biol Phys 2006;64:784–791.

findings. Semin Oncol 2002;29:38–41.

dysfunction. Int J Radiat Oncol Biol Phys 1994;29:747–754.

homogenates. Int J Radiat Oncol Biol Phys 1984;10:1529–1531.

2721 before fractionated irradiation of the head and neck to reduce salivary gland

tumor bearing mice by s-2-(3-aminopropylamino)- ethylphosphorothioic acid (WR

prevent radiation-induced proctosigmoiditis. A phase I/II trial. Cancer

small-cell lung cancer patients receiving chemotherapy and hyperfractionated radiation: Radiation Therapy Oncology Group trial 98-01. J Clin Oncol

of head and neck tumors measured by quantitative pertechnetate scintigraphy: Comparison of intensity-modulated radiotherapy and conventional radiation therapy with and without amifostine. Int J Radiat Oncol Biol Phys 2007;67:651–659.

neck tumors undergoing radio-chemotherapy. Is it possible to limit the duration of

2721 and its thiol, WR-1065: Implications for mechanisms of radioprotection. Int J

response rate during radiotherapy: Results of a meta-analysis. Int J Radiat Oncol

metabolites of the drug ethyol (WR-2721), protect DNA against fast neutron-

lung cancer patients receiving concurrent carboplatin, paclitaxel, and radiation therapy followed by gemcitabine and cisplatin intensification: Preliminary

Cancer 1992;69:2820–2825.

1992;69:2826–2830.

2005;23:2145–2154.

2721). Am J Clin Oncol 1982;5:321–328.


Glover D, Riley L, Carmichael K et al. Hypocalcemia and inhibition of parathyroid hormone

Hensley ML, Hagerty KL, Kewalramani T, et al. American Society of Clinical Oncology 2008

Hensley ML, Schuchter LM, Lindley C et al. American Society of Clinical Oncology clinical

Jellema AP, Slotman BJ, Muller MJ, et al. Radiotherapy alone, versus radiotherapy with

Kajstura J, Cheng W, Reiss K et al. Apoptotic and necrotic myocyte cell deaths are

Kligerman MM, Liu T, Liu Y, Scheffler B, He S, Zhang Z. Interim analysis of a randomized

Komaki R, Lee JS, Milas L, et al. Effects of amifostine on acute toxicity from concurrent

radiotherapy-induced esophagitis. Clin Cancer Res 1999;5:3970–3976. Koukourakis MI, Kyrias G, Kakolyris S et al. Subcutaneous administration of amifostine

Koukourakis MI. Amifostine: Is there evidence of tumor protection? Semin Oncol

Kouloulias VE, Kouvaris JR, Pissakas G et al. A phase II randomized study of topical

Kouloulias VE, Kouvaris JR, Pissakas G et al. Phase II multicenter randomized study of

Kouvaris J, Kouloulias V, Malas E et al. Amifostine as radioprotective agent for the rectal

Leong SS, Tan EH, Fong K, et al. Randomized double-blind trial of combined modality

induced rectal toxicity. Strahlenther Onkol 2004;180:557–562.

retrospective analysis. Onkologie. 2002 Aug;25(4):364-9.

cancer. J Clin Oncol 2003;21:1767–1774.

agent). N Engl J Med 1983;309:1137–1141.

Clin Oncol 1999;17:3333–3355.

2006 Aug 1;107(3):544-53.

Biol Phys. 1992;22(4):799-802

2000;18:2226–2233.

2003;30(suppl 18):18–30.

protectants. J Clin Oncol. 2009 Jan 1;27(1):127-45.

secretion after administration of WR-2721 (a radioprotective and chemoprotective

clinical practice guideline update: use of chemotherapy and radiation therapy

practice guidelines for the use of chemotherapy and radiotherapy protectants. J

amifostine 3 times weekly, versus radiotherapy with amifostine 5 times weekly: A prospective randomized study in squamous cell head and neck cancer. Cancer.

independent contributing variables of infarct size in rats. Lab Invest 1996;74:86–107.

trial of radiation therapy of rectal cancer with/without WR-2721. Int J Radiat Oncol

chemotherapy and radiotherapy for inoperable non-small-cell lung cancer: report of a randomized comparative trial. Int J Radiat Oncol Biol Phys. 2004;58(5):1369-77 Koukourakis MI, Flordellis CS, Giatromanolaki A et al. Oral administration of recombinant

human granulocyte macrophage colony-stimulating factor in the management of

during fractionated radiotherapy: A randomized phase II study. J Clin Oncol

intrarectal administration of amifostine for the prevention of acute radiation-

amifostine for prevention of acute radiation rectal toxicity: Topical intrarectal versus subcutaneous application. Int J Radiat Oncol Biol Phys 2005;62:486–493. Kouvaris J, Kouloulias V, Kokakis J et al. The cytoprotective effect of amifostine in acute radiation dermatitis: A retrospective analysis. Eur J Dermatol 2002;12:458–462. Kouvaris J, Kouloulias V, Kokakis J, Matsopoulos G, Balafouta M, Miliadou A, Vlahos L.

Cytoprotective effect of amifostine in radiation-induced acute mucositis - a

mucosa during irradiation of pelvic tumors. A phase II randomized study using various toxicity scales and rectosigmoidoscopy. Strahlenther Onkol 2003;179:167–174.

treatment with or without amifostine in unresectable stage III non-small-cell lung


**15** 

*Japan* 

Kenshiro Shiraishi *Department of Radiology,* 

 *The University of Tokyo Hospital,* 

**Abscopal Effect of Radiation Therapy:** 

**Current Concepts and Future Applications** 

Radiation therapy is one of the most important treatment tools in cancer therapy. It has a wide variety of indications for many malignant tumors, mostly for local control, whether a curative or palliative outcome is the intent, or as pre- or post-operative treatment as either neoadjuvant or adjuvant therapy. Radiation therapy is commonly used along with hormone therapy or chemotherapy. The full scope of the capabilities of radiation therapy is achieved particularly in combination settings with various anti-tumor modalities, the so-called multidisciplinary approach. To enhance the therapeutic efficacy of radiation sufficiently, one may choose radiation therapy in combination with cytotoxic chemotherapeutic agents or with warming devices used for hyperthermia treatment or utilize newly developing physical approaches as typified by intensity modulated radiation therapy, stereotactic radiation therapy, brachytherapy and image-guided radiation therapy. Moreover, an immunoenhancing agent might be selected in combination with radiation therapy from the standpoint of immunobiology in the treatment of cancer. Some promising data have been shown on the basis of immunological activation with ionizing radiation, demonstrating cytotoxic T lymphocyte (CTL) amplification and dendritic cell (DC) stimulation or maturation (Demaria, et al., 2004,Ganss, et al., 2002,Nikitina and Gabrilovich, 2001,Schuler,

Radiation therapy plays a crucial role in enhancing tumor immunogenicity by promoting cross-priming and eliciting anti-tumor T-cell responses, and generates inflammatory signals via induction of tumor cell death (Hong, et al., 1999,Quarmby, et al., 1999,Watters, 1999). Thus, ionizing radiation can achieve not only direct cancer cell death but also has an indirect and systemic anti-tumor mechanism outside of the irradiated field, which has been reported in some clinical settings (Antoniades, et al., 1977,Ehlers and Fridman, 1973,Kingsley, 1975,Nobler, 1969,Perego and Faravelli, 2000,Rees, 1981,Rees and Ross, 1983,Sham, 1995). Local irradiation resulted in an anti-tumor effect at a non-irradiated location in a patient with hepatocellular carcinoma that regressed after palliative local radiotherapy for pain control of bone metastases (Ohba, et al., 1998). This rare phenomenon is known as the abscopal effect and is defined as a reaction following irradiation but occurring outside the zone of actual radiation absorption (Mole, 1953). The word "abscopal" is derived from the Latin prefix "ab," meaning "away from," and the Greek word "scopos," meaning "target."

**1. Introduction** 

et al., 2003).


## **Abscopal Effect of Radiation Therapy: Current Concepts and Future Applications**

Kenshiro Shiraishi

*Department of Radiology, The University of Tokyo Hospital, Japan* 

#### **1. Introduction**

274 Modern Practices in Radiation Therapy

Shaw L, Brown W, Schein P et al. A phase I study comparing the relative bioavailability of

Shaw LM, Turrisi AT, Glover DJ et al. Human pharmacokinetics of WR-2721. Int J Radiat

Shimizu S, Eguchi Y, Kamiike W et al. Induction of apoptosis as well as necrosis by hypoxia

Simone N, Menard C, Singh A. Intrarectal amifostine suspension may protect against acute

Singh AK, Menard C, Guion P et al. Intrarectal amifostine suspension may protect against

Sonis ST, Fey EG. Oral complications of cancer therapy. Oncology (Williston Park)

Stone HB, Coleman CN, Anscher MS et al. Effects of radiation on normal tissue:

Travis EL. The oxygen dependence of protection by aminothiols: Implications for normal tissues and solid tumors. Int J Radiat Oncol Biol Phys 1984;10:1495–1501. Utley JF, Seaver N, Newton GL et al. Pharmacokinetics of WR-1065 in mouse tissue

Vacha P, Fehlauer F, Mahlmann B et al. Randomized phase III trial of postoperative

Wagner W, Prott FJ, Schonekas KG. Amifostine: A radioprotector in locally advanced head

Washburn LC, Rafter JJ, Hayes RL. Prediction of the effective radioprotective dose of WR-

Wasserman TH, Brizel DM, Henke M et al. Influence of intravenous amifostine on

Yuhas JM, Spellman JM, Culo F. The role of WR-2721 in radiotherapy and/or

Yuhas JM, Storer JB. Differential chemoprotection of normal and malignant tissues. J Natl

Yuhas JM. Active versus passive absorption kinetics as the basis for selective protection of

Consequences and mechanisms. Lancet Oncol 2003;4:529–536.

radioprotection? Strahlenther Onkol 2003;179:385–389.

and neck tumors. Oncol Rep 1998;5:1255–1257.

chemotherapy. Cancer Clin Trials 1980;3:211–216.

Proc Am Soc Clin Oncol 1997;16:250a.

Radiat Oncol Biol Phys 2006;65:1008–1013.

2002;16:680–686, 691–692, 695; discussion 686.

Oncol Biol Phys 1986;12:1501–1504.

1996;56:2161–2166.

Chicago, IL, 2005.

1976;66:100–105.

Oncol Biol Phys 2005;63:985–990.

Cancer Inst 1969;42:331–335.

Res 1980;40:1519–1524.

intravenous (i.v.) and subcutaneous (s.c.) administration of amifostine (Ethyol).

and predominant prevention of apoptosis by Bcl-2 and Bcl-XL. Cancer Res

proctitis during prostate radiation therapy: A pilot study. Presented at the Radiological Society of North America Annual Meeting; November 27–December 2;

acute proctitis during radiation therapy for prostate cancer: A pilot study. Int J

following treatment with WR-2721. Int J Radiat Oncol Biol Phys 1984;10:1525–1528.

radiochemotherapy +/- amifostine in head and neck cancer. Is there evidence for

2721 in humans through an interspecies tissue distribution study. Radiat Res

xerostomia, tumor control, and survival after radiotherapy for head-and-neck cancer: 2-year follow-up of a prospective, randomized, phase III trial. Int J Radiat

normal tissues by S-2-(3-aminopropylamino)-ethylphosphorothioic acid. Cancer

Radiation therapy is one of the most important treatment tools in cancer therapy. It has a wide variety of indications for many malignant tumors, mostly for local control, whether a curative or palliative outcome is the intent, or as pre- or post-operative treatment as either neoadjuvant or adjuvant therapy. Radiation therapy is commonly used along with hormone therapy or chemotherapy. The full scope of the capabilities of radiation therapy is achieved particularly in combination settings with various anti-tumor modalities, the so-called multidisciplinary approach. To enhance the therapeutic efficacy of radiation sufficiently, one may choose radiation therapy in combination with cytotoxic chemotherapeutic agents or with warming devices used for hyperthermia treatment or utilize newly developing physical approaches as typified by intensity modulated radiation therapy, stereotactic radiation therapy, brachytherapy and image-guided radiation therapy. Moreover, an immunoenhancing agent might be selected in combination with radiation therapy from the standpoint of immunobiology in the treatment of cancer. Some promising data have been shown on the basis of immunological activation with ionizing radiation, demonstrating cytotoxic T lymphocyte (CTL) amplification and dendritic cell (DC) stimulation or maturation (Demaria, et al., 2004,Ganss, et al., 2002,Nikitina and Gabrilovich, 2001,Schuler, et al., 2003).

Radiation therapy plays a crucial role in enhancing tumor immunogenicity by promoting cross-priming and eliciting anti-tumor T-cell responses, and generates inflammatory signals via induction of tumor cell death (Hong, et al., 1999,Quarmby, et al., 1999,Watters, 1999). Thus, ionizing radiation can achieve not only direct cancer cell death but also has an indirect and systemic anti-tumor mechanism outside of the irradiated field, which has been reported in some clinical settings (Antoniades, et al., 1977,Ehlers and Fridman, 1973,Kingsley, 1975,Nobler, 1969,Perego and Faravelli, 2000,Rees, 1981,Rees and Ross, 1983,Sham, 1995). Local irradiation resulted in an anti-tumor effect at a non-irradiated location in a patient with hepatocellular carcinoma that regressed after palliative local radiotherapy for pain control of bone metastases (Ohba, et al., 1998). This rare phenomenon is known as the abscopal effect and is defined as a reaction following irradiation but occurring outside the zone of actual radiation absorption (Mole, 1953). The word "abscopal" is derived from the Latin prefix "ab," meaning "away from," and the Greek word "scopos," meaning "target."

Abscopal Effect of Radiation Therapy: Current Concepts and Future Applications 277

including palpation, indicated an abscopal effect on metastatic lymph nodes in 15 out of 42 cases (35.7%). Pathologic findings revealed an even greater tendency for regression, with an abscopal effect demonstrated in tissue samples from 22 of 42 cases (52.4%). Thus, more than half of these patients with advanced breast cancer exhibited some sort of abscopal effect following irradiation and surgery. The incidence of the abscopal effect was significantly higher in patients under 55 years old and was most frequent in patients who had "infiltrating lymphocytes around the degenerated cancer cells in the irradiated primary tumor nests." In other words, under the favorable condition of a vigorous immune reaction to the tumor as indicated by the presence of abundant lymphocytes, the host was more likely to attack the tumor and bring about an abscopal response as a result. Among the types of lymphocytes, the authors claimed that the most prevalent cells had been identified as primarily CD8 and CD4 lymphocytes, which play a role in cellular defense against pathogens, malignant cells, and other foreign substances. According to the authors, their findings suggested that the abscopal effect was caused by activated cellular immunity in the hosts. Although the study was not large enough for data to yield statistically significant results, the survival rate among patients who exhibited the abscopal effect was higher than

The logical inference from this research is that the abscopal effect is a desirable and common systemic reaction to localized cancer treatment. Since the abscopal effect is dependent on a healthy immune system, one might infer that immune-damaging treatments should be kept to a minimum. In terms of this point, the trend in most parts of the world is in the undesirable direction, and immunosuppressive chemotherapy is given at every opportunity. The recruitment of leukocytes may have been inhibited by the antitumor chemotherapeutic agents, which would support the assumption that some types of recruited leukocytes play a

Blay *et al.* reported that higher pretreatment interleukin (IL)-6 and C-reactive protein (CRP) levels in renal cell carcinoma were associated with a diminished response to cytokine therapy and poorer survival. Survival appeared to be better in those patients that had elevated CRP values that decreased to normal levels after nephrectomy compared to those whose CRP did not decrease to normal. For those whose pre-treatment CRP was within normal limits, there was no difference in survival between those who did or did not undergo nephrectomy (Blay, et al., 1992). Fujikawa *et al.* proposed that an IL-6-induced inflammatory response might inhibit the immune anti-tumor response. They suggested the following mechanism: in the setting of metastatic renal cell carcinoma and a primary tumor predominantly expressing IL-6, an associated drop in CRP following nephrectomy appears to curb the inflammatory response while simultaneously inducing immune activation

role in the enhancement of the efficacy of radiation and the abscopal effect.

**3. Basic research for induction of radiation-related abscopal effect** 

Fms-like tyrosine kinase receptor 3 ligand (Flt3-L) is a growth factor that stimulates production of DCs and has been shown to induce antitumor immunity to several mouse

**3.1 Basic research on the basis of immunological mechanisms** 

among those patients who showed no such reaction.

**2.4 Surgery-related abscopal effect** 

(Fujikawa, et al., 2000).

The abscopal mechanism of action remains to be clarified, although a variety of underlying biological events can be hypothesized, mainly those induced by the immune system (Macklis, et al., 1992,Uchida, et al., 1989). Thus far, immunological activation with local irradiation has been explained by multiple possible mechanisms (Awwad and North, 1990,Cameron, et al., 1990,Chiang, et al., 1997,Dybal, et al., 1992,Younes, et al., 1995,Younes, et al., 1995).

This chapter gives an overview of theoretical mechanisms of the abscopal effect being progressively elucidated in the development of multidisciplinary approaches for cancer therapy.

#### **2. Speculation on the mechanism of the abscopal effect**

#### **2.1 Possible cytokine contribution**

Historically, the abscopal effect has been described in various tumors with possible underlying mechanisms explaining each observed case. A 76-year-old patient with hepatocellular carcinoma was irradiated to control his bone metastases as palliative, not curative, therapy. Yet following this palliative radiotherapy the primary liver tumor regressed (Ohba, et al., 1998). Ohba *et al.* also found in this patient an increase in blood levels of tumor necrosis factor alpha (TNF-), which has known anti-tumor activity. They suggested that the primary tumor regression might have been caused by an immune response spearheaded by TNF-. TNF has a paradoxical role in cancer by promoting growth, invasion, and metastasis in some tumors, while having a reverse effect in other cancers through destruction of blood vessels and cell-mediated killing. One wonderful review of the relation between TNF- and cancer is found in the Lancet Oncology (Szlosarek and Balkwill, 2003).

#### **2.2 Hyperthermia-related abscopal effect**

Abscopal effects are usually associated with radiation therapy, however, one could sometimes see after other treatments as well, such as surgery or even hyperthermia. For example, in an experiment conducted in India, administering hyperthermia to the hind leg of a mouse for 40 min before transplanting a fibrosarcoma reduced the growth of the tumor in the heated leg (Vartak, et al., 1993). More surprisingly, it inhibited the growth of a tumor transplanted to the unheated leg as well. Actually, two to three weeks after hyperthermic treatment, tumor growth retardation had ceased in the leg that had been heated, but was still noticeable in the leg that had not been heated. Although the mechanism for this effect had not been investigated, the abscopal effect from hyperthermia turned out to be greater than its direct effect on the local target tumor. The authors concluded that local hyperthermia induced both direct and abscopal anti-tumor effects that might probably be the result of a systemic effect of hyperthermia in the host animal.

#### **2.3 Radiation-related abscopal effect**

In the clinical setting, Konoeda *et al.* conducted a practical study to investigate the mechanism of the abscopal effect in patients with breast cancer (Konoeda, 1990). Study subjects were 62 women with advanced breast cancer who received radiation therapy before surgery and then underwent mastectomy or tumor resection. Physical examination, including palpation, indicated an abscopal effect on metastatic lymph nodes in 15 out of 42 cases (35.7%). Pathologic findings revealed an even greater tendency for regression, with an abscopal effect demonstrated in tissue samples from 22 of 42 cases (52.4%). Thus, more than half of these patients with advanced breast cancer exhibited some sort of abscopal effect following irradiation and surgery. The incidence of the abscopal effect was significantly higher in patients under 55 years old and was most frequent in patients who had "infiltrating lymphocytes around the degenerated cancer cells in the irradiated primary tumor nests." In other words, under the favorable condition of a vigorous immune reaction to the tumor as indicated by the presence of abundant lymphocytes, the host was more likely to attack the tumor and bring about an abscopal response as a result. Among the types of lymphocytes, the authors claimed that the most prevalent cells had been identified as primarily CD8 and CD4 lymphocytes, which play a role in cellular defense against pathogens, malignant cells, and other foreign substances. According to the authors, their findings suggested that the abscopal effect was caused by activated cellular immunity in the hosts. Although the study was not large enough for data to yield statistically significant results, the survival rate among patients who exhibited the abscopal effect was higher than among those patients who showed no such reaction.

The logical inference from this research is that the abscopal effect is a desirable and common systemic reaction to localized cancer treatment. Since the abscopal effect is dependent on a healthy immune system, one might infer that immune-damaging treatments should be kept to a minimum. In terms of this point, the trend in most parts of the world is in the undesirable direction, and immunosuppressive chemotherapy is given at every opportunity. The recruitment of leukocytes may have been inhibited by the antitumor chemotherapeutic agents, which would support the assumption that some types of recruited leukocytes play a role in the enhancement of the efficacy of radiation and the abscopal effect.

#### **2.4 Surgery-related abscopal effect**

276 Modern Practices in Radiation Therapy

The abscopal mechanism of action remains to be clarified, although a variety of underlying biological events can be hypothesized, mainly those induced by the immune system (Macklis, et al., 1992,Uchida, et al., 1989). Thus far, immunological activation with local irradiation has been explained by multiple possible mechanisms (Awwad and North, 1990,Cameron, et al., 1990,Chiang, et al., 1997,Dybal, et al., 1992,Younes, et al., 1995,Younes,

This chapter gives an overview of theoretical mechanisms of the abscopal effect being progressively elucidated in the development of multidisciplinary approaches for cancer

Historically, the abscopal effect has been described in various tumors with possible underlying mechanisms explaining each observed case. A 76-year-old patient with hepatocellular carcinoma was irradiated to control his bone metastases as palliative, not curative, therapy. Yet following this palliative radiotherapy the primary liver tumor regressed (Ohba, et al., 1998). Ohba *et al.* also found in this patient an increase in blood levels of tumor necrosis factor alpha (TNF-), which has known anti-tumor activity. They suggested that the primary tumor regression might have been caused by an immune response spearheaded by TNF-. TNF has a paradoxical role in cancer by promoting growth, invasion, and metastasis in some tumors, while having a reverse effect in other cancers through destruction of blood vessels and cell-mediated killing. One wonderful review of the relation between TNF- and cancer is

Abscopal effects are usually associated with radiation therapy, however, one could sometimes see after other treatments as well, such as surgery or even hyperthermia. For example, in an experiment conducted in India, administering hyperthermia to the hind leg of a mouse for 40 min before transplanting a fibrosarcoma reduced the growth of the tumor in the heated leg (Vartak, et al., 1993). More surprisingly, it inhibited the growth of a tumor transplanted to the unheated leg as well. Actually, two to three weeks after hyperthermic treatment, tumor growth retardation had ceased in the leg that had been heated, but was still noticeable in the leg that had not been heated. Although the mechanism for this effect had not been investigated, the abscopal effect from hyperthermia turned out to be greater than its direct effect on the local target tumor. The authors concluded that local hyperthermia induced both direct and abscopal anti-tumor effects that might probably be

In the clinical setting, Konoeda *et al.* conducted a practical study to investigate the mechanism of the abscopal effect in patients with breast cancer (Konoeda, 1990). Study subjects were 62 women with advanced breast cancer who received radiation therapy before surgery and then underwent mastectomy or tumor resection. Physical examination,

**2. Speculation on the mechanism of the abscopal effect** 

found in the Lancet Oncology (Szlosarek and Balkwill, 2003).

the result of a systemic effect of hyperthermia in the host animal.

**2.2 Hyperthermia-related abscopal effect** 

**2.3 Radiation-related abscopal effect** 

**2.1 Possible cytokine contribution** 

et al., 1995).

therapy.

Blay *et al.* reported that higher pretreatment interleukin (IL)-6 and C-reactive protein (CRP) levels in renal cell carcinoma were associated with a diminished response to cytokine therapy and poorer survival. Survival appeared to be better in those patients that had elevated CRP values that decreased to normal levels after nephrectomy compared to those whose CRP did not decrease to normal. For those whose pre-treatment CRP was within normal limits, there was no difference in survival between those who did or did not undergo nephrectomy (Blay, et al., 1992). Fujikawa *et al.* proposed that an IL-6-induced inflammatory response might inhibit the immune anti-tumor response. They suggested the following mechanism: in the setting of metastatic renal cell carcinoma and a primary tumor predominantly expressing IL-6, an associated drop in CRP following nephrectomy appears to curb the inflammatory response while simultaneously inducing immune activation (Fujikawa, et al., 2000).

#### **3. Basic research for induction of radiation-related abscopal effect**

#### **3.1 Basic research on the basis of immunological mechanisms**

Fms-like tyrosine kinase receptor 3 ligand (Flt3-L) is a growth factor that stimulates production of DCs and has been shown to induce antitumor immunity to several mouse

Abscopal Effect of Radiation Therapy: Current Concepts and Future Applications 279

Important factor is that radiation therapy appears to cause less immunosuppression compared to surgery or other invasive treatment modalities. Therefore, radiation therapy potentially should have the more favorable biological activity for inducing an abscopal effect than surgical procedures if the major underlying mechanism is based upon immune

The abscopal effect apparently operates through mechanisms that are paralleled in gene therapy, local immunotherapy, hyperthermia, and post-surgical distant bystander effects. Recently, some investigators have suggested that the definition of the abscopal effect should have been broadened to include other forms of local therapy that have systemic effects, *i.e.*, a distant bystander effect (Perego and Faravelli, 2000,Vartak, et al., 1993). Whether or not the definition should be extended to include local therapies other than radiation therapy that have a distant effect is a matter of debate. However, to unravel the abscopal effect of radiation, it seems prudent to evaluate other directed therapies that are associated with systemic effects (Kaminski, et al., 2005). Since the literal meaning is the same for abscopal and distant bystander, the terms could be used interchangeably to refer to any local therapy

In recent years, the crucial role played by innate immunity, and in particular by DCs in enhancing T cell activation, has been widely clarified. DCs are lineage-negative, bone marrow-derived mononuclear cells found in peripheral blood or in many organs (O'Neill, et al., 2004). DCs can be broadly divided into myeloid or plasmacytoid DCs (MDCs and PDCs, respectively) on the basis of phenotypic, morphologic, and functional differences. Antigens acquired both endogenously (*i.e.*, synthesized within the DC cytosol) or exogenously (acquired from the extracellular environment) are processed into peptides, which are loaded onto major histocompatibility complex class I and II (MHC I and II) molecules and transported to the cell surface for recognition by antigen-specific T cells. DCs most efficiently capture antigens when they are in the immature phase. The terminal process of differentiation termed as *maturation* transforms DCs with weak immunostimulatory properties for antigen capture into cells specialized for T cell stimulation in the lymphoid organ. This process is accompanied by cytoskeletal reorganization, loss of adhesiveness, acquisition of cellular motility with development of characteristic cytoplasmic extensions, migration to lymphoid tissues, reduced phagocytic uptake, and T cell activation potential (O'Neill, et al., 2004). Natural killer (NK) cells are activated by type I interferon (IFN) produced from tumor tissues as a "danger signal" to attack tumor cells. Immature DCs uptake tumor tissue-derived products such as apoptotic bodies and necrotic bodies with tumor-associated antigens (Moretta, 2002). Mature DCs can secrete chemokines and cytokines that attract other immune cells and activate resting T cells. DCs can prime resting NK cells via proinflammatory cytokines such as IL-12 or IL-15 and NK-inducing chemokines such as IL-8 or macrophage inflammatory protein 1-alpha (MIP-1), and enhance their own maturation by attachment with activated NK cells. However, NK cells negatively regulate the function of DCs also by killing immature DCs in peripheral tissues. Moreover, a subset of NK cells, after migration to secondary lymphoid tissues, might have a role in the editing of mature DCs based on the selective killing of mature DCs that do not express optimal surface densities of MHC class I molecules. Maturation of DCs can be

activation.

with a distant impact.

**3.2 Possible mechanisms via DC activation** 

tumors, although its effects as a single agent are limited to early and more immunogenic tumors (Maraskovsky, et al., 1996,Maraskovsky, et al., 1997). The first study to test the combination of Flt3-L with local irradiation used the Lewis lung model of metastatic carcinoma (Chakravarty, et al., 1999). When Flt3-L was administered after the ablation of the primary tumor by high-dose local irradiation with 60 Gy, lung metastasis formation was inhibited and disease-free survival was enhanced compared with that of mice treated with irradiation or Flt3-L alone. Importantly, the anti-metastatic effect required T cells because this effect was not observed in nude (T cell-deficient) mice. These results provide preliminary evidence in support of the hypothesis that radiation-induced tumor cell death can release antigens for DCs to present to T cells. The high single dose of radiation used in this study limits its clinical applicability in addition to the fact that the intrinsic tumor immunogenicity could explain these responses. Nevertheless, these studies provided initial proof of the principle and stimulated some groups to further investigate whether more clinically relevant radiation doses could be used to elicit systemic antitumor immunity in combination with Flt3-L.

Demaria *et al.* used mouse mammary carcinoma 67NR, a moderately immunogenic syngeneic tumor. A radiation dose sufficient to cause growth delay of the irradiated tumor, in this case 2 Gy, was able to induce a systemic antitumor effect only in combination with Flt3-L administration. Inhibition of tumor growth outside of the irradiated field was specific and required T cells, confirming that it was immune-mediated (Demaria, et al., 2004).

Other groups have used a slightly different approach based on the same hypothesis, that radiation can free tumor-derived antigens for DC uptake and presentation. Nikitina *et al.* used *in vitro* bone marrow-derived DCs that were injected i.v. and s.c. around the tumor after local irradiation (Nikitina and Gabrilovich, 2001) whereas Teitz-Tennenbaum *et al.*  used intratumoral injection of DCs (Teitz-Tennenbaum, et al., 2003). In both cases, the administration of DCs after radiation therapy was able to induce a potent antitumor immune response. Yasuda *et al.* reported intratumoral IL-2 injection after irradiation to colon adenocarcinoma enhances antitumor local control and abscopal metastatic inhibition via CD4 positive lymphocytes (Yasuda, et al., 2011). In another study, p53 appeared to mediate a radiation-induced abscopal effect in mice that was dose dependent (Camphausen, et al., 2003). Table 1 summarizes the possible underlying mechanisms for the abscopal effects observed preclinically or clinically.


Table 1. Possible mechanisms for the abscopal effect

Important factor is that radiation therapy appears to cause less immunosuppression compared to surgery or other invasive treatment modalities. Therefore, radiation therapy potentially should have the more favorable biological activity for inducing an abscopal effect than surgical procedures if the major underlying mechanism is based upon immune activation.

The abscopal effect apparently operates through mechanisms that are paralleled in gene therapy, local immunotherapy, hyperthermia, and post-surgical distant bystander effects. Recently, some investigators have suggested that the definition of the abscopal effect should have been broadened to include other forms of local therapy that have systemic effects, *i.e.*, a distant bystander effect (Perego and Faravelli, 2000,Vartak, et al., 1993). Whether or not the definition should be extended to include local therapies other than radiation therapy that have a distant effect is a matter of debate. However, to unravel the abscopal effect of radiation, it seems prudent to evaluate other directed therapies that are associated with systemic effects (Kaminski, et al., 2005). Since the literal meaning is the same for abscopal and distant bystander, the terms could be used interchangeably to refer to any local therapy with a distant impact.

#### **3.2 Possible mechanisms via DC activation**

278 Modern Practices in Radiation Therapy

tumors, although its effects as a single agent are limited to early and more immunogenic tumors (Maraskovsky, et al., 1996,Maraskovsky, et al., 1997). The first study to test the combination of Flt3-L with local irradiation used the Lewis lung model of metastatic carcinoma (Chakravarty, et al., 1999). When Flt3-L was administered after the ablation of the primary tumor by high-dose local irradiation with 60 Gy, lung metastasis formation was inhibited and disease-free survival was enhanced compared with that of mice treated with irradiation or Flt3-L alone. Importantly, the anti-metastatic effect required T cells because this effect was not observed in nude (T cell-deficient) mice. These results provide preliminary evidence in support of the hypothesis that radiation-induced tumor cell death can release antigens for DCs to present to T cells. The high single dose of radiation used in this study limits its clinical applicability in addition to the fact that the intrinsic tumor immunogenicity could explain these responses. Nevertheless, these studies provided initial proof of the principle and stimulated some groups to further investigate whether more clinically relevant radiation doses could be used to elicit systemic antitumor immunity in

Demaria *et al.* used mouse mammary carcinoma 67NR, a moderately immunogenic syngeneic tumor. A radiation dose sufficient to cause growth delay of the irradiated tumor, in this case 2 Gy, was able to induce a systemic antitumor effect only in combination with Flt3-L administration. Inhibition of tumor growth outside of the irradiated field was specific and required T cells, confirming that it was immune-mediated (Demaria, et al., 2004).

Other groups have used a slightly different approach based on the same hypothesis, that radiation can free tumor-derived antigens for DC uptake and presentation. Nikitina *et al.* used *in vitro* bone marrow-derived DCs that were injected i.v. and s.c. around the tumor after local irradiation (Nikitina and Gabrilovich, 2001) whereas Teitz-Tennenbaum *et al.*  used intratumoral injection of DCs (Teitz-Tennenbaum, et al., 2003). In both cases, the administration of DCs after radiation therapy was able to induce a potent antitumor immune response. Yasuda *et al.* reported intratumoral IL-2 injection after irradiation to colon adenocarcinoma enhances antitumor local control and abscopal metastatic inhibition via CD4 positive lymphocytes (Yasuda, et al., 2011). In another study, p53 appeared to mediate a radiation-induced abscopal effect in mice that was dose dependent (Camphausen, et al., 2003). Table 1 summarizes the possible underlying mechanisms for the abscopal

Author Tumor type Treated sites (treatment) Observed abscopal effect Putative intrinsic mediator that induces abscopal effect

Table 1. Type of malignancies in relation to abscopal effect reported and possible underlying mechanism.

Abbreviations: RT, radiation therapy; HT, hyperthermia; RFA, radiofrequency ablation; LLC, Lewis lung carcinoma; DC ,dendritic cells; NK, natural killer; TNF, tumor necrosis factor; IL, interleukin; CRP, C-reactive prot

Vartak *et al*. fibrosarcoma hind leg (HT) tumor growth inhibition of unheated leg unknown Chakravarty *et al*. LLC primary tumor (RT) lung metastasis regression DC Demaria *et al*. mammary carcinoma 67NR primary tumor (RT) distant tumor growth inhibition DC Teitz-Tennenbaum *et al*. melanoma/sarcoma primary tumor (RT) lung metastasis regression DC Camphausen *et al*. LLC/fibrosarcoma hind leg (RT) distant tumor growth inhibition p53

Iida *et al*. hepatocellular carcinoma primary tumor (RFA) distant tumor growth inhibition DC Yasuda *et al*. colon adenocarcinoma primary tumor (RT) liver metastasis inhibition CD4 lymphocytes

Ohba *et al*. hepatocellular carcinoma bone metastasis (RT) primary tumor regression TNF-a

Shiraishi *et al*. colon adenocarcinoma/LLC/fibrosarcoma primary tumor (RT) distant tumor growth inhibition/longer survival CD8 and CD4 lymphocytes/NK

Konoeda *et al*. breast cancer breast (RT) metastatic lymph node regression CD8 and CD4 lymphocytes Blay *et al*. renal cell cacinoma nephrectomy (surgery) longer survival IL-6 and CRP Fujikawa *et al*. metastatic renal cell carcinoma nephrectomy (surgery) longer survival IL-6

combination with Flt3-L.

effects observed preclinically or clinically.

*Preclinical*

*Clinical*

Table 1. Possible mechanisms for the abscopal effect

In recent years, the crucial role played by innate immunity, and in particular by DCs in enhancing T cell activation, has been widely clarified. DCs are lineage-negative, bone marrow-derived mononuclear cells found in peripheral blood or in many organs (O'Neill, et al., 2004). DCs can be broadly divided into myeloid or plasmacytoid DCs (MDCs and PDCs, respectively) on the basis of phenotypic, morphologic, and functional differences. Antigens acquired both endogenously (*i.e.*, synthesized within the DC cytosol) or exogenously (acquired from the extracellular environment) are processed into peptides, which are loaded onto major histocompatibility complex class I and II (MHC I and II) molecules and transported to the cell surface for recognition by antigen-specific T cells. DCs most efficiently capture antigens when they are in the immature phase. The terminal process of differentiation termed as *maturation* transforms DCs with weak immunostimulatory properties for antigen capture into cells specialized for T cell stimulation in the lymphoid organ. This process is accompanied by cytoskeletal reorganization, loss of adhesiveness, acquisition of cellular motility with development of characteristic cytoplasmic extensions, migration to lymphoid tissues, reduced phagocytic uptake, and T cell activation potential (O'Neill, et al., 2004). Natural killer (NK) cells are activated by type I interferon (IFN) produced from tumor tissues as a "danger signal" to attack tumor cells. Immature DCs uptake tumor tissue-derived products such as apoptotic bodies and necrotic bodies with tumor-associated antigens (Moretta, 2002). Mature DCs can secrete chemokines and cytokines that attract other immune cells and activate resting T cells. DCs can prime resting NK cells via proinflammatory cytokines such as IL-12 or IL-15 and NK-inducing chemokines such as IL-8 or macrophage inflammatory protein 1-alpha (MIP-1), and enhance their own maturation by attachment with activated NK cells. However, NK cells negatively regulate the function of DCs also by killing immature DCs in peripheral tissues. Moreover, a subset of NK cells, after migration to secondary lymphoid tissues, might have a role in the editing of mature DCs based on the selective killing of mature DCs that do not express optimal surface densities of MHC class I molecules. Maturation of DCs can be

Abscopal Effect of Radiation Therapy: Current Concepts and Future Applications 281

Deventer, et al., 2002), enhancement of radiation efficacy had not been investigated sufficiently. Radiation treatment at tumor bearing sites is known to induce strong inflammation in the irradiated field and to recruit tumor-specific T lymphocytes and DCs, which seem to play an important role in the remission of tumors (Friedman, 2002,Garnett, et al., 2004,Teitz-Tennenbaum, et al., 2003). MIP-1 or CCL3, is a chemokine known to be secreted from various leukocytes including T lymphocytes and activated macrophages, and to recruit CCR1- and/or CCR5-expressing leukocytes such as monocytes, DCs, NK cells and T lymphocytes (Rollins, 1997). It was also reported that MIP-1 could enhance survival of DCs (Sumida, et al., 2004) and primed T lymphocytes to generate IFN-

An active variant of human MIP-1 with improved pharmaceutical properties that carries a single amino acid substitution of the 26Asp to Ala was reported (Hunter, et al., 1995), which has a reduced tendency to form large aggregates at physiological pH and ionic strength. Myelosuppressive effect of the active variant (Arango, et al., 1999,Arango, et al., 2001,Gilmore, et al., 1999,Lord, et al., 1995) was investigated in several clinical trials of patients receiving chemotherapy (Bernstein, et al., 1997,Broxmeyer, et al., 1998,Clemons, et al., 1998,Marshall, et al., 1998). We previously showed that the recombinant MIP-1 variant, now called ECI301, strikingly enhanced the antitumor efficacy of subcutaneous tumor irradiation and induced an abscopal effect (Shiraishi, et al., 2008). Our study resulted in tumor-free mice with long-term survival without significant toxicity and complete rejection by surviving mice to a re-challenge with the same tumor cells. In accordance with our findings, no significant side effects of a compound with the same structure (BB-10010) had been reported previously when administered to human patients. Moreover, we observed a tumor-type- and mouse-strain-independent abscopal effect, indicating that the antitumor effect of ECI301 may be exerted via systemic inflammation and immune response. Marked infiltration of CD4+ and CD8+ cells was observed both in irradiated and non-irradiated sites. It was reported that DC precursors were mobilized into the circulation by administration of MIP-1 (Zhang, et al., 2004) and radiofrequency ablation-treated hepatocellular carcinomas (Iida, et al., 2010); however, we did not observe an increase in CD11c+ cell infiltration into the tumor tissue in this model. Depletion of CD8+ T cells by antibodies diminished the effect of combination treatment at the irradiated site, indicating that CD8+ T cells are involved in the antitumor effect. Furthermore, rejection of the same tumor type in the cured mice may have been mediated by the presence of these types of lymphocytes. An increased number of splenocytes with tumor-specific IFN--generating ability with the combination treatment also supports this assumption (Shiraishi, et al., 2010). Depletion of CD4+ T lymphocytes or NK1.1 cells by antibodies diminished the abscopal effect, indicating that these cells are involved in the remission either directly or indirectly. CD4+ T cells may play a role in generating cytokines such as IFN-, which may also activate other leukocytes (Dorner, et al.,

Further studies using C3H/HeN, C3H/HeJ and athymic mice will show whether the high mobility group box 1 (HMGB1) RNA level, an important mediator of local and systemic inflammation, is up-regulated at each tumor-bearing site (unpublished data). Results might clarify the underlying HMGB1-dependent mechanism for the abscopal effect via TLR4-

(Lillard, et al., 2003).

2002,Pender, et al., 2005,Shiraishi, et al., 2008).

mediated inflammation (Fig. 1).

induced by a growing number of exogenous and endogenous molecular signals, generally referred to as "danger signals" (Matzinger, 1994). Danger signals include host-derived proinflammatory cytokines, such as TNF, IL-1, IL-6, and type I IFN, and a variety of molecules released not only by microbes but also by damaged host tissues, including tumor involvement (Skoberne, et al., 2004). These noncytokine molecules signal primarily through transmembrane receptors related to Drosophila Toll protein, known as Toll-like receptors (TLR) (Kopp and Medzhitov, 2003), which are expressed by DCs.

The major concern is whether ionizing radiation-induced apoptosis can increase tumor immunogenicity. The immunostimulatory activity associated with cell lysates (endogenous adjuvant activity) was shown to be elevated once the cells were stressed by ultraviolet radiation, indicating that injury can modulate this effect (Gallucci, et al., 1999,Shi, et al., 2000). Some examples exist in which apoptotic cells show immunostimulatory features (Rock, et al., 2005). Immunization with apoptotic cells or *in situ* induction of tumor cell apoptosis induced T cell responses *in vivo* as exemplified in some reports (Kotera, et al., 2001,Nowak, et al., 2003,Ronchetti, et al., 1999). Injection of immature DCs into tumor tissue after irradiation-induced tumor cell apoptosis can stimulate strong antitumor immunity (Kim, et al., 2004). These studies suggest that under some favorable conditions for an immunocompetent host, radiation-induced tumor cell death might be associated with the production of ideal maturation signals for DCs (Demaria, et al., 2005).

The possible contribution of radiation-induced apoptosis vs. necrosis to immunostimulation has not been fully elucidated, and no significant difference was seen in capabilities of both kinds of cell death for antigen presentation *in vitro* (Larsson, et al., 2001). Endogenous factors released from necrotic cells might be responsible for the ability of the necrotic body to activate DCs (Skoberne, et al., 2004). Examples of these are immunostimulatory self-DNA that binds TLR9, self-single-strand RNA that stimulates TLR7 and TLR8, secondary structures of messenger RNA that activate TLR3, and heat shock proteins that stimulate TLR4 (Demaria, et al., 2005). The induction of necrosis *in vivo* could be accompanied by the release not only of self-antigens but also inflammatory factors that may cause DC maturation and the whole immune response. Candidates for cell-associated antigens being cross-presented from dying cells could include heat shock protein-associated proteins, native proteins (Shen and Rock, 2004), peptides (Neijssen, et al., 2005), or other constituents. In general, it is considered that DC maturation signals are essential to convert crosstolerance to cross-priming (Steinman and Nussenzweig, 2002).

Opinion is divided as to the ability of ionizing radiation to generate the signals required for DC maturation; however, the combined approach of inducing cell death by irradiation in combination with the administration of a chemotactic agent that activates DCs can lead to the priming or enhancement of antitumor responses (Shiraishi, et al., 2008).

#### **3.3 Attempts to consistently induce the abscopal effect**

Based on the theory of immunological activation with ionizing radiation, Shiraishi *et al.* have chosen MIP-1 in combination with radiotherapy and investigated whether MIP-1 could cause a broad-spectrum enhancement of the efficacy of radiotherapy in tumor-bearing mice. Although there are many reports concerning anti-cancer (Crittenden, et al., 2003,Nakashima, et al., 1996,Taub, et al., 1995,Zibert, et al., 2004) and anti-metastasis effects of MIP-1 (van

induced by a growing number of exogenous and endogenous molecular signals, generally referred to as "danger signals" (Matzinger, 1994). Danger signals include host-derived proinflammatory cytokines, such as TNF, IL-1, IL-6, and type I IFN, and a variety of molecules released not only by microbes but also by damaged host tissues, including tumor involvement (Skoberne, et al., 2004). These noncytokine molecules signal primarily through transmembrane receptors related to Drosophila Toll protein, known as Toll-like receptors

The major concern is whether ionizing radiation-induced apoptosis can increase tumor immunogenicity. The immunostimulatory activity associated with cell lysates (endogenous adjuvant activity) was shown to be elevated once the cells were stressed by ultraviolet radiation, indicating that injury can modulate this effect (Gallucci, et al., 1999,Shi, et al., 2000). Some examples exist in which apoptotic cells show immunostimulatory features (Rock, et al., 2005). Immunization with apoptotic cells or *in situ* induction of tumor cell apoptosis induced T cell responses *in vivo* as exemplified in some reports (Kotera, et al., 2001,Nowak, et al., 2003,Ronchetti, et al., 1999). Injection of immature DCs into tumor tissue after irradiation-induced tumor cell apoptosis can stimulate strong antitumor immunity (Kim, et al., 2004). These studies suggest that under some favorable conditions for an immunocompetent host, radiation-induced tumor cell death might be associated with the

The possible contribution of radiation-induced apoptosis vs. necrosis to immunostimulation has not been fully elucidated, and no significant difference was seen in capabilities of both kinds of cell death for antigen presentation *in vitro* (Larsson, et al., 2001). Endogenous factors released from necrotic cells might be responsible for the ability of the necrotic body to activate DCs (Skoberne, et al., 2004). Examples of these are immunostimulatory self-DNA that binds TLR9, self-single-strand RNA that stimulates TLR7 and TLR8, secondary structures of messenger RNA that activate TLR3, and heat shock proteins that stimulate TLR4 (Demaria, et al., 2005). The induction of necrosis *in vivo* could be accompanied by the release not only of self-antigens but also inflammatory factors that may cause DC maturation and the whole immune response. Candidates for cell-associated antigens being cross-presented from dying cells could include heat shock protein-associated proteins, native proteins (Shen and Rock, 2004), peptides (Neijssen, et al., 2005), or other constituents. In general, it is considered that DC maturation signals are essential to convert cross-

Opinion is divided as to the ability of ionizing radiation to generate the signals required for DC maturation; however, the combined approach of inducing cell death by irradiation in combination with the administration of a chemotactic agent that activates DCs can lead to

Based on the theory of immunological activation with ionizing radiation, Shiraishi *et al.* have chosen MIP-1 in combination with radiotherapy and investigated whether MIP-1 could cause a broad-spectrum enhancement of the efficacy of radiotherapy in tumor-bearing mice. Although there are many reports concerning anti-cancer (Crittenden, et al., 2003,Nakashima, et al., 1996,Taub, et al., 1995,Zibert, et al., 2004) and anti-metastasis effects of MIP-1 (van

(TLR) (Kopp and Medzhitov, 2003), which are expressed by DCs.

production of ideal maturation signals for DCs (Demaria, et al., 2005).

tolerance to cross-priming (Steinman and Nussenzweig, 2002).

**3.3 Attempts to consistently induce the abscopal effect** 

the priming or enhancement of antitumor responses (Shiraishi, et al., 2008).

Deventer, et al., 2002), enhancement of radiation efficacy had not been investigated sufficiently. Radiation treatment at tumor bearing sites is known to induce strong inflammation in the irradiated field and to recruit tumor-specific T lymphocytes and DCs, which seem to play an important role in the remission of tumors (Friedman, 2002,Garnett, et al., 2004,Teitz-Tennenbaum, et al., 2003). MIP-1 or CCL3, is a chemokine known to be secreted from various leukocytes including T lymphocytes and activated macrophages, and to recruit CCR1- and/or CCR5-expressing leukocytes such as monocytes, DCs, NK cells and T lymphocytes (Rollins, 1997). It was also reported that MIP-1 could enhance survival of DCs (Sumida, et al., 2004) and primed T lymphocytes to generate IFN- (Lillard, et al., 2003).

An active variant of human MIP-1 with improved pharmaceutical properties that carries a single amino acid substitution of the 26Asp to Ala was reported (Hunter, et al., 1995), which has a reduced tendency to form large aggregates at physiological pH and ionic strength. Myelosuppressive effect of the active variant (Arango, et al., 1999,Arango, et al., 2001,Gilmore, et al., 1999,Lord, et al., 1995) was investigated in several clinical trials of patients receiving chemotherapy (Bernstein, et al., 1997,Broxmeyer, et al., 1998,Clemons, et al., 1998,Marshall, et al., 1998). We previously showed that the recombinant MIP-1 variant, now called ECI301, strikingly enhanced the antitumor efficacy of subcutaneous tumor irradiation and induced an abscopal effect (Shiraishi, et al., 2008). Our study resulted in tumor-free mice with long-term survival without significant toxicity and complete rejection by surviving mice to a re-challenge with the same tumor cells. In accordance with our findings, no significant side effects of a compound with the same structure (BB-10010) had been reported previously when administered to human patients. Moreover, we observed a tumor-type- and mouse-strain-independent abscopal effect, indicating that the antitumor effect of ECI301 may be exerted via systemic inflammation and immune response. Marked infiltration of CD4+ and CD8+ cells was observed both in irradiated and non-irradiated sites. It was reported that DC precursors were mobilized into the circulation by administration of MIP-1 (Zhang, et al., 2004) and radiofrequency ablation-treated hepatocellular carcinomas (Iida, et al., 2010); however, we did not observe an increase in CD11c+ cell infiltration into the tumor tissue in this model. Depletion of CD8+ T cells by antibodies diminished the effect of combination treatment at the irradiated site, indicating that CD8+ T cells are involved in the antitumor effect. Furthermore, rejection of the same tumor type in the cured mice may have been mediated by the presence of these types of lymphocytes. An increased number of splenocytes with tumor-specific IFN--generating ability with the combination treatment also supports this assumption (Shiraishi, et al., 2010). Depletion of CD4+ T lymphocytes or NK1.1 cells by antibodies diminished the abscopal effect, indicating that these cells are involved in the remission either directly or indirectly. CD4+ T cells may play a role in generating cytokines such as IFN-, which may also activate other leukocytes (Dorner, et al., 2002,Pender, et al., 2005,Shiraishi, et al., 2008).

Further studies using C3H/HeN, C3H/HeJ and athymic mice will show whether the high mobility group box 1 (HMGB1) RNA level, an important mediator of local and systemic inflammation, is up-regulated at each tumor-bearing site (unpublished data). Results might clarify the underlying HMGB1-dependent mechanism for the abscopal effect via TLR4 mediated inflammation (Fig. 1).

Abscopal Effect of Radiation Therapy: Current Concepts and Future Applications 283

In conclusion, data that possibly support an intriguing concept as an abscopal effect are reviewed. These data will encourage future therapeutic gain of immunostimulants utilization in the treatment of advanced or metastatic cancer. The development of safer, reasonable, and targeted therapies will be facilitated as we clarify the mechanisms for the abscopal effects. Future therapies will need to be optimized with tumor-type tailoring in consideration of various intra- or inter-tissue signals if these are to affect treatment outcome. Hopefully, a more aggressive effort for investigating and developing a potentially novel application of ionizing radiation in combination with immunotherapy will be needed. When the effectiveness of "immunoradiotherapy" in a clinical setting is established in a desirable manner, it could lead to a new era of cancer treatment, with common availability of

**5. List of abbreviations and expansions in the order corresponding to** 

Antoniades *et al.* (1977). Lymphangiographic demonstration of the abscopal effect in

Arango *et al.* (1999). BB-10010, an analogue of macrophage inflammatory protein-1 alpha,

Arango *et al.* (2001). BB-10010, an analog of macrophage inflammatory protein-1alpha,

Awwad & North. (1990). Radiosensitive barrier to T-cell-mediated adoptive immunotherapy

Bernstein *et al.* (1997). A randomized phase II study of BB-10010: a variant of human

patients with malignant lymphomas. *Int J Radiat Oncol Biol Phys*, Vol.2, No.1-2, pp.

reduces proliferation in murine small-intestinal crypts. *Scand J Gastroenterol*, Vol.34,

protects murine small intestine against radiation. *Dig Dis Sci*, Vol.46, No.12, pp.

of established tumors. *Cancer Res*, Vol.50, No.8, pp. 2228-2233, ISSN 0008-5472

macrophage inflammatory protein-1alpha for patients receiving high-dose

established modalities, without significant adverse events.

**4. Conclusion**

**apperances** 

DC, dendritic cell

NK, natural killer IFN, interferon

**6. References**

CRP, C-reactive protein

TLR, Toll-like receptors

(Print)

IL , interleukin

CTL, cytotoxic T lymphocyte

TNF-, tumor necrosis factor alpha

HMGB1, high mobility group box 1

Flt3-L, fms-like tyrosine kinase receptor 3 MHC, major histocompatibility complex

MIP-1, macrophage inflammatory protein 1-alpha

141-147, ISSN 0360-3016 (Print)

No.1, pp. 68-72, ISSN 0036-5521 (Print)

2608-2614, ISSN 0163-2116 (Print)

Fig. 1. Possible mechanism for radiation-induced abscopal effect.

Ionizing radiation induces tumor cell death in the irradiated tumor, causes inflammation and activates the immune system via chemokines with HMGB1. Length of arrows means relative strength of the effects.

HMGB1 = high mobility group box 13.4 Implications for future therapies

For future development, further insights into the mechanisms underpinning abscopal signaling are required. Theoretical elucidation of the relevance of abscopal responses in radiation-induced carcinogenesis is also required, including molecular pathways and targets outside of directly exposed fields.

A balance between angiogenic and anti-angiogenic molecules seems to be one of the key factors behind tumor growth. For example, several experimental animal models indeed suggest that the growth of a primary tumor can inhibit the production of distant metastases, probably due to inhibition of angiogenesis (Gorelik, 1983,Prehn, 1991). In contrast, the angiogenic inhibitors, angiostatin and endostatin, are known to function in tumor inhibition (O'Reilly, et al., 1997,O'Reilly, et al., 1994). Hartford *et al.* reported that the effect of irradiation of a primary tumor on angiogenesis at a distal site may differ from the effect of surgical removal of the primary tumor with respect to angiostatin production (Hartford, et al., 2000). They clearly demonstrated that, unlike surgery, irradiation of a tumor might enhance angiogenic suppression at a distal site. The involvement of angiogenic regulation in a radiation-induced abscopal effect should be emphasized as a clinical advantage in contrast to other invasive procedures, which may reduce possible angiogenic inhibition.

### **4. Conclusion**

282 Modern Practices in Radiation Therapy

Fig. 1. Possible mechanism for radiation-induced abscopal effect.

HMGB1 = high mobility group box 13.4 Implications for future therapies

relative strength of the effects.

outside of directly exposed fields.

Ionizing radiation induces tumor cell death in the irradiated tumor, causes inflammation and activates the immune system via chemokines with HMGB1. Length of arrows means

For future development, further insights into the mechanisms underpinning abscopal signaling are required. Theoretical elucidation of the relevance of abscopal responses in radiation-induced carcinogenesis is also required, including molecular pathways and targets

A balance between angiogenic and anti-angiogenic molecules seems to be one of the key factors behind tumor growth. For example, several experimental animal models indeed suggest that the growth of a primary tumor can inhibit the production of distant metastases, probably due to inhibition of angiogenesis (Gorelik, 1983,Prehn, 1991). In contrast, the angiogenic inhibitors, angiostatin and endostatin, are known to function in tumor inhibition (O'Reilly, et al., 1997,O'Reilly, et al., 1994). Hartford *et al.* reported that the effect of irradiation of a primary tumor on angiogenesis at a distal site may differ from the effect of surgical removal of the primary tumor with respect to angiostatin production (Hartford, et al., 2000). They clearly demonstrated that, unlike surgery, irradiation of a tumor might enhance angiogenic suppression at a distal site. The involvement of angiogenic regulation in a radiation-induced abscopal effect should be emphasized as a clinical advantage in contrast

to other invasive procedures, which may reduce possible angiogenic inhibition.

In conclusion, data that possibly support an intriguing concept as an abscopal effect are reviewed. These data will encourage future therapeutic gain of immunostimulants utilization in the treatment of advanced or metastatic cancer. The development of safer, reasonable, and targeted therapies will be facilitated as we clarify the mechanisms for the abscopal effects. Future therapies will need to be optimized with tumor-type tailoring in consideration of various intra- or inter-tissue signals if these are to affect treatment outcome.

Hopefully, a more aggressive effort for investigating and developing a potentially novel application of ionizing radiation in combination with immunotherapy will be needed. When the effectiveness of "immunoradiotherapy" in a clinical setting is established in a desirable manner, it could lead to a new era of cancer treatment, with common availability of established modalities, without significant adverse events.

#### **5. List of abbreviations and expansions in the order corresponding to apperances**

CTL, cytotoxic T lymphocyte DC, dendritic cell TNF-, tumor necrosis factor alpha IL , interleukin CRP, C-reactive protein Flt3-L, fms-like tyrosine kinase receptor 3 MHC, major histocompatibility complex NK, natural killer IFN, interferon MIP-1, macrophage inflammatory protein 1-alpha TLR, Toll-like receptors HMGB1, high mobility group box 1

#### **6. References**


Abscopal Effect of Radiation Therapy: Current Concepts and Future Applications 285

Gallucci *et al.* (1999). Natural adjuvants: endogenous activators of dendritic cells. *Nat Med*, Vol.5, No.11, pp. 1249-1255, ISSN 1078-8956 (Print) 1078-8956 (Linking) Ganss *et al.* (2002). Combination of T-cell therapy and trigger of inflammation induces

Garnett *et al.* (2004). Sublethal irradiation of human tumor cells modulates phenotype

Gilmore *et al.* (1999). Protective effects of BB-10010 treatment on chemotherapy-induced

Gorelik. (1983). Concomitant tumor immunity and the resistance to a second tumor

Hartford *et al.* (2000). Irradiation of a Primary Tumor, Unlike Surgical Removal, Enhances

Hong *et al.* (1999). Rapid induction of cytokine gene expression in the lung after single and

Hunter *et al.* (1995). BB-10010: an active variant of human macrophage inflammatory

Iida *et al.* (2010). Antitumor effect after radiofrequency ablation of murine hepatoma is

Kaminski *et al.* (2005). The controversial abscopal effect. *Cancer Treat Rev*, Vol.31, No.3, pp.

Kim *et al.* (2004). Direct injection of immature dendritic cells into irradiated tumor induces

Kingsley. (1975). An interesting case of possible abscopal effect in malignant melanoma. *Br J* 

Konoeda. (1990). [Therapeutic efficacy of pre-operative radiotherapy on breast carcinoma: in

Kotera *et al.* (2001). Comparative analysis of necrotic and apoptotic tumor cells as a source of

Larsson *et al.* (2001). Efficiency of cross presentation of vaccinia virus-derived antigens by

Lillard *et al.* (2003). MIP-1alpha and MIP-1beta differentially mediate mucosal and systemic adaptive immunity. *Blood*, Vol.101, No.3, pp. 807-814, ISSN 0006-4971 (Print)

*Chiryo Gakkai Shi*, Vol.25, No.6, pp. 1204-1214, ISSN 0021-4671 (Print) Kopp & Medzhitov. (2003). Recognition of microbial infection by Toll-like receptors. *Curr* 

*Radiol*, Vol.48, No.574, pp. 863-866, ISSN 0007-1285 (Print)

8109, ISSN 0008-5472 (Print) 0008-5472 (Linking)

Interaction. *Cancer Res*, Vol.60, No.8, pp. 2128-2131, ISSN

1462-1470, ISSN 0008-5472 (Print)

4400-4408, ISSN 0006-4971 (Print)

7445 (Electronic) 0008-5472 (Linking)

159-172, ISSN 0305-7372 (Print)

7136 (Print) 0020-7136 (Linking)

(Print) 0014-2980 (Linking)

(Linking)

0955-3002 (Print)

pp. 7985-7994, ISSN 0008-5472 (Print)

remodeling of the vasculature and tumor eradication. *Cancer Res*, Vol.62, No.5, pp.

resulting in enhanced killing by cytotoxic T lymphocytes. *Cancer Res*, Vol.64, No.21,

neutropenia in mice. *Exp Hematol*, Vol.27, No.2, pp. 195-202, ISSN 0301-472X (Print)

challenge. *Adv Cancer Res*, Vol.39, pp. 71-120, ISSN 0065-230X (Print) 0065-230X

Angiogenesis Suppression at a Distal Site: Potential Role of Host-Tumor

fractionated doses of radiation. *Int J Radiat Biol*, Vol.75, No.11, pp. 1421-1427, ISSN

protein-1 alpha with improved pharmaceutical properties. *Blood*, Vol.86, No.12, pp.

augmented by an active variant of CC Chemokine ligand 3/macrophage inflammatory protein-1alpha. *Cancer Res*, Vol.70, No.16, pp. 6556-6565, ISSN 1538-

efficient antitumor immunity. *Int J Cancer*, Vol.109, No.5, pp. 685-690, ISSN 0020-

special reference to its abscopal effect on metastatic lymph-nodes]. *Nippon Gan* 

*Opin Immunol*, Vol.15, No.4, pp. 396-401, ISSN 0952-7915 (Print) 0952-7915 (Linking)

antigen(s) in dendritic cell-based immunization. *Cancer Res*, Vol.61, No.22, pp. 8105-

human dendritic cells. *Eur J Immunol*, Vol.31, No.12, pp. 3432-3442, ISSN 0014-2980

etoposide and cyclophosphamide for malignant lymphoma and breast cancer. *Br J Haematol*, Vol.99, No.4, pp. 888-895, ISSN 0007-1048 (Print)


Blay *et al.* (1992). Serum level of interleukin 6 as a prognosis factor in metastatic renal cell

Broxmeyer *et al.* (1998). Myeloid progenitor cell proliferation and mobilization effects of

cancer. *Blood Cells Mol Dis*, Vol.24, No.1, pp. 14-30, ISSN 1079-9796 (Print) Cameron *et al.* (1990). Synergistic antitumor activity of tumor-infiltrating lymphocytes,

Camphausen *et al.* (2003). Radiation abscopal antitumor effect is mediated through p53.

Chakravarty *et al.* (1999). Flt3-ligand administration after radiation therapy prolongs

Chiang *et al.* (1997). Effects of IL-3 gene expression on tumor response to irradiation in vitro and in vivo. *Cancer Res*, Vol.57, No.18, pp. 3899-3903, ISSN 0008-5472 (Print) Clemons *et al.* (1998). A randomized phase-II study of BB-10010 (macrophage inflammatory

Crittenden *et al.* (2003). Expression of inflammatory chemokines combined with local tumor

Demaria *et al.* (2005). Combining radiotherapy and immunotherapy: a revived partnership.

Demaria *et al.* (2004). Ionizing radiation inhibition of distant untreated tumors (abscopal

Dorner *et al.* (2002). MIP-1alpha, MIP-1beta, RANTES, and ATAC/lymphotactin function

Dybal *et al.* (1992). Synergy of radiation therapy and immunotherapy in murine renal cell carcinoma. *J Urol*, Vol.148, No.4, pp. 1331-1337, ISSN 0022-5347 (Print) Ehlers & Fridman. (1973). Abscopal effect of radiation in papillary adenocarcinoma. *Br J* 

Friedman. (2002). Immune modulation by ionizing radiation and its implications for cancer

Fujikawa *et al.* (2000). Serum immunosuppressive acidic protein and natural killer cell

*Haematol*, Vol.99, No.4, pp. 888-895, ISSN 0007-1048 (Print)

*Med*, Vol.171, No.1, pp. 249-263, ISSN 0022-1007 (Print)

6028-6032, ISSN 0008-5472 (Print) 0008-5472 (Linking)

Vol.63, No.17, pp. 5505-5512, ISSN 0008-5472 (Print)

No.9, pp. 6181-6186, ISSN 0027-8424 (Print)

*Radiol*, Vol.46, No.543, pp. 220-222, ISSN 0007-1285 (Print)

1540, ISSN 0006-4971 (Print)

3016 (Linking)

ISSN 0360-3016 (Print)

(Print) 1381-6128 (Linking)

5347 (Linking)

*Cancer Res*, Vol.63, No.8, pp. 1990-1993, ISSN 0008-5472 (Print)

5472 (Linking)

etoposide and cyclophosphamide for malignant lymphoma and breast cancer. *Br J* 

carcinoma. *Cancer Res*, Vol.52, No.12, pp. 3317-3322, ISSN 0008-5472 (Print) 0008-

BB10010, a genetically engineered variant of human macrophage inflammatory protein-1alpha, in a phase I clinical trial in patients with relapsed/refractory breast

interleukin 2, and local tumor irradiation. Studies on the mechanism of action. *J Exp* 

survival in a murine model of metastatic lung cancer. *Cancer Res*, Vol.59, No.24, pp.

protein- 1alpha) in patients with advanced breast cancer receiving 5-fluorouracil, adriamycin, and cyclophosphamide chemotherapy. *Blood*, Vol.92, No.5, pp. 1532-

destruction enhances tumor regression and long-term immunity. *Cancer Res*,

*Int J Radiat Oncol Biol Phys*, Vol.63, No.3, pp. 655-666, ISSN 0360-3016 (Print) 0360-

effect) is immune mediated. *Int J Radiat Oncol Biol Phys*, Vol.58, No.3, pp. 862-870,

together with IFN-gamma as type 1 cytokines. *Proc Natl Acad Sci U S A*, Vol.99,

immunotherapy. *Curr Pharm Des*, Vol.8, No.19, pp. 1765-1780, ISSN 1381-6128

activity in patients with metastatic renal cell carcinoma before and after nephrectomy. *J Urol*, Vol.164, No.3 Pt 1, pp. 673-675, ISSN 0022-5347 (Print) 0022-


Abscopal Effect of Radiation Therapy: Current Concepts and Future Applications 287

O'Reilly *et al.* (1994). Angiostatin: a novel angiogenesis inhibitor that mediates the

Ohba *et al.* (1998). Abscopal regression of hepatocellular carcinoma after radiotherapy for bone metastasis. *Gut*, Vol.43, No.4, pp. 575-577, ISSN 0017-5749 (Print) Pender *et al.* (2005). Systemic administration of the chemokine macrophage inflammatory

Perego & Faravelli. (2000). Unexpected consequence of splenectomy in composite

Prehn. (1991). The Inhibition of Tumor Growth by Tumor Mass. *Cancer Res*, Vol.51, No.1, pp.

Quarmby *et al.* (1999). Radiation-induced normal tissue injury: role of adhesion molecules in

Rees. (1981). Abscopal regression in lymphoma: a mechanism in common with total body irradiation? *Clin Radiol*, Vol.32, No.4, pp. 475-480, ISSN 0009-9260 (Print) Rees & Ross. (1983). Abscopal regression following radiotherapy for adenocarcinoma. *Br J* 

Rock *et al.* (2005). Natural endogenous adjuvants. *Springer Semin Immunopathol*, Vol.26, No.3,

Ronchetti *et al.* (1999). Immunogenicity of apoptotic cells in vivo: role of antigen load,

Schuler *et al.* (2003). The use of dendritic cells in cancer immunotherapy. *Curr Opin Immunol*,

Sham. (1995). The abscopal effect and chronic lymphocytic leukemia. *Am J Med*, Vol.98,

Shen & Rock. (2004). Cellular protein is the source of cross-priming antigen in vivo. *Proc* 

Shi *et al.* (2000). Cell injury releases endogenous adjuvants that stimulate cytotoxic T cell

Shiraishi *et al.* (2008). Enhancement of antitumor radiation efficacy and consistent induction

Shiraishi *et al.* (2010). Enhancement of antitumor radiation efficacy and the abscopal effect

*American Association for Cancer Research 101st Annual Meeting 2010.* p. 5617 Skoberne *et al.* (2004). Danger signals: a time and space continuum. *Trends Mol Med*, Vol.10,

No.6, pp. 251-257, ISSN 1471-4914 (Print) 1471-4914 (Linking)

antigen-presenting cells, and cytokines. *J Immunol*, Vol.163, No.1, pp. 130-136, ISSN

*Natl Acad Sci U S A*, Vol.101, No.9, pp. 3035-3040, ISSN 0027-8424 (Print) 0027-8424

responses. *Proc Natl Acad Sci U S A*, Vol.97, No.26, pp. 14590-14595, ISSN 0027-8424

of the abscopal effect in mice by ECI301, an active variant of macrophage inflammatory protein-1alpha. *Clin Cancer Res*, Vol.14, No.4, pp. 1159-1166, ISSN

by ECI301 mediated TLR4 dependent innate immunity in mice, *Proceedings of*

Rollins. (1997). Chemokines. *Blood*, Vol.90, No.3, pp. 909-928, ISSN 0006-4971 (Print)

328, ISSN 0092-8674 (Print) 0092-8674 (Linking)

Vol.54, No.8, pp. 1114-1120, ISSN 0017-5749 (Print)

*Radiol*, Vol.56, No.661, pp. 63-66, ISSN 0007-1285 (Print)

pp. 231-246, ISSN 0344-4325 (Print) 0344-4325 (Linking)

0022-1767 (Print) 0022-1767 (Linking)

No.3, pp. 307-308, ISSN 0002-9343 (Print)

Vol.15, No.2, pp. 138-147, ISSN 0952-7915 (Print)

6078 (Print)

0020-7136 (Print)

2-4, ISSN

(Linking)

(Print) 0027-8424 (Linking)

1078-0432 (Print)

suppression of metastases by a Lewis lung carcinoma. *Cell*, Vol.79, No.2, pp. 315-

protein 1alpha exacerbates inflammatory bowel disease in a mouse model. *Gut*,

lymphoma. The abscopal effect. *Haematologica*, Vol.85, No.2, pp. 211, ISSN 0390-

leukocyte-endothelial cell interactions. *Int J Cancer*, Vol.82, No.3, pp. 385-395, ISSN


Lord *et al.* (1995). Mobilization of early hematopoietic progenitor cells with BB-10010: a

Macklis *et al.* (1992). Lymphoid irradiation results in long-term increases in natural killer

Maraskovsky *et al.* (1996). Dramatic increase in the numbers of functionally mature dendritic

Marshall *et al.* (1998). Clinical effects of human macrophage inflammatory protein-1 alpha

Matzinger. (1994). Tolerance, danger, and the extended family. *Annu Rev Immunol*, Vol.12,

Mole. (1953). Whole body irradiation; radiobiology or medicine? *Br J Radiol*, Vol.26, No.305,

Moretta. (2002). Natural killer cells and dendritic cells: rendezvous in abused tissues. *Nat Rev Immunol*, Vol.2, No.12, pp. 957-964, ISSN 1474-1733 (Print) 1474-1733 (Linking) Nakashima *et al.* (1996). A candidate for cancer gene therapy: MIP-1 alpha gene transfer to

Neijssen *et al.* (2005). Cross-presentation by intercellular peptide transfer through gap

Nikitina & Gabrilovich. (2001). Combination of gamma-irradiation and dendritic cell

Nobler. (1969). The abscopal effect in malignant lymphoma and its relationship to

Nowak *et al.* (2003). Induction of tumor cell apoptosis in vivo increases tumor antigen cross-

O'Neill *et al.* (2004). Manipulating dendritic cell biology for the active immunotherapy of

O'Reilly *et al.* (1997). Endostatin: an endogenous inhibitor of angiogenesis and tumor growth. *Cell*, Vol.88, No.2, pp. 277-285, ISSN 0092-8674 (Print) 0092-8674 (Linking)

*Eur J Cancer*, Vol.34, No.7, pp. 1023-1029, ISSN 0959-8049 (Print)

pp. 991-1045, ISSN 0732-0582 (Print) 0732-0582 (Linking)

alpha. *Blood*, Vol.85, No.12, pp. 3412-3415, ISSN 0006-4971 (Print)

ISSN 0008-543X (Print)

(Print) 0065-2598 (Linking)

pp. 234-241, ISSN 0007-1285 (Print)

ISSN 0724-8741 (Print)

833, ISSN 0020-7136 (Print)

4687 (Linking)

(Linking)

(Linking)

genetically engineered variant of human macrophage inflammatory protein-1

cells in patients treated for Hodgkin's disease. *Cancer*, Vol.69, No.3, pp. 778-783,

cells in Flt3 ligand-treated mice: multiple dendritic cell subpopulations identified. *J Exp Med*, Vol.184, No.5, pp. 1953-1962, ISSN 0022-1007 (Print) 0022-1007 (Linking) Maraskovsky *et al.* (1997). Dramatic numerical increase of functionally mature dendritic cells

in FLT3 ligand-treated mice. *Adv Exp Med Biol*, Vol.417, pp. 33-40, ISSN 0065-2598

MIP-1 alpha (LD78) administration to humans: a phase I study in cancer patients and normal healthy volunteers with the genetically engineered variant, BB-10010.

an adenocarcinoma cell line reduced tumorigenicity and induced protective immunity in immunocompetent mice. *Pharm Res*, Vol.13, No.12, pp. 1896-1901,

junctions. *Nature*, Vol.434, No.7029, pp. 83-88, ISSN 1476-4687 (Electronic) 1476-

administration induces a potent antitumor response in tumor-bearing mice: approach to treatment of advanced stage cancer. *Int J Cancer*, Vol.94, No.6, pp. 825-

lymphocyte circulation. *Radiology*, Vol.93, No.2, pp. 410-412, ISSN 0033-8419 (Print)

presentation, cross-priming rather than cross-tolerizing host tumor-specific CD8 T cells. *J Immunol*, Vol.170, No.10, pp. 4905-4913, ISSN 0022-1767 (Print) 0022-1767

cancer. *Blood*, Vol.104, No.8, pp. 2235-2246, ISSN 0006-4971 (Print) 0006-4971


**Part 6** 

**Emerging Dosimeters and New QA Practices** 

