11.6 Translational Research in Radiation Oncology of Lung Cancer

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11.6 Translational Research in Radiation Oncology of Lung Cancer

Yuhchyau Chen

Y. Chen, PhD, MD

Associate Professor, Director of Clinical Investigation, Department of Radiation Oncology, 601 Elmwood Ave., Box 647, Rochester, NY 14642, USA

CONTENTS

11.6.1 Introduction 481

11.6.2 Optimizing Paclitaxel Chemoradiation Sensitizing Strategy in Stage III

Non-Small Cell Lung Cancer: A Phase I/II Study Based on Pre-Clinical Research 481 11.6.2.1 Paclitaxel Chemoradiation for NSCLC 482 11.6.2.2 Pharmacokinetic Consideration

of Paclitaxel for Radiosensitization 483 11.6.2.3 Paclitaxel Cell Cycle Effect on Human

Cancer Cell Lines 484 11.6.2.4 Paclitaxel Apoptotic Effect 484 11.6.2.5 Schedule-Dependent Radiosensitizing Effect In Vitro 484

11.6.2.6 Clinical Protocol of Pulsed Paclitaxel and Radiation 485

11.6.2.7 Tumor Response, In-Field Tumor Control, and Survival 485

11.6.2.8 Toxicity 486

11.6.2.9 Future Directions in Chemopotentiation of Radiotherapy for Stage III NSCLC 487 11.6.3 Cytokine Markers for Radiation

Pulmonary Injury 487 11.6.3.1 Radiation Lung Injury 487

11.6.3.2 Cytokine and Radiation Lung Injury 488 11.6.3.3 Inﬂ ammatory Cytokines IL-1α and IL-6 488 11.6.3.4 Chemokines and Cell Adhesion Molecules 489 11.6.3.5 Fibrotic Cytokine: Transforming

Growth Factor β 490 11.6.4 Conclusions 490 References 491

11.6.1 Introduction

Although radiation is an essential modality for cancer therapy because of its powerful DNA-damaging effects, those same effects unfortunately cause simultaneous injury to patients’ normal tissues. Thus the success of radiotherapy relies on the ﬁ ne bal- ance between eradicating cancer cells and minimiz-

ing injury to normal tissues. In lung cancer research and treatment, basic biomedical research and clinical studies have ﬂ ourished in the past two decades, but lung cancer remains the most deadly cancer of all types. Despite the explosion of discoveries that have elucidated our understanding of the molecular mechanisms and regulatory signals involved in cancer progression, transferring knowledge into practi- cal advances for treating lung cancer and preventing side effects from cytotoxic therapy has been a more deliberate process. The translation of knowledge gained from the laboratory into improving treatment outcomes will serve as the vital bridge between sci- entiﬁ c discovery and the welfare of patients and will offer hope for further improvement of lung cancer therapy.

Because the scope of this chapter is limited, it will focus on two areas of translational research in radiation oncology for lung cancer treatment: 1) the attempt to potentiate radiation effects by a unique radiosensitizing strategy that optimally integrates paclitaxel and radiation to enhance radiation’s local effects in treating locally advanced non-small cell lung cancer (NSCLC), and 2) the cytokines involved in radiation lung injury that lead to radiation pneumonitis and progressive ﬁ brosis.

11.6.2

Optimizing Paclitaxel Chemoradiation Sensitizing Strategy in Stage III Non-Small Cell Lung Cancer: A Phase I/II Study Based on Pre-Clinical Research

The treatment of locally advanced, stage III NSCLC remains a challenging task for oncologists because of lack of local disease control and high rates of distant disease failures. Although aggressive combination chemoradiation therapy is now the new standard of treatment for stage III NSCLC, 5-year survival rates are only in the range of 5%–25%, and local failure rates have been as high as 55%–85% (Dillman et

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al. 1990; Sause et al. 2000; Schaake-Koning et al.

1992; Arriagada et al. 1991; Morton et al. 1991).

In addition, due to aggravated toxicity from therapy, combined modality treatment is offered to a limited population of patients in good clinical performance status only. Both the optimal regimen and sequence of chemoradiation therapy for stage III NSCLC remain unclear.

At least two randomized studies have demonstrated that concurrent chemoradiation is more effective than sequential chemotherapy followed by radiation for the treatment of stage III NSCLC, sup- porting the radiosensitization mechanism in improving survival of lung cancer patients (Furuse et al. 1999; Curran et al. 2000). The phase III ran- domized study conducted by the Radiation Therapy Oncology Group (RTOG) for stage III NSCLC represents a good example of the dilemma in trading toxicity for efﬁ cacy. RTOG 94–10 compared three arms of chemoradiation treatments: sequential chemoradiation vs. concurrent chemotherapy and once-daily (QD) radiation vs. concurrent chemotherapy and twice-daily (BID) radiation. The combined chemoradiation strategy resulted in a modest survival gain in the concurrent chemoradiation arm, with a median survival of 17 months for the concurrent QD arm vs.

14.6 months for the sequential chemoradiation arm (P =.038), and a median survival of 15.6 months for the concurrent BID arm. Most notable was a dramatic increase in grade 3/4 acute nonhematologic toxicities such as esophagitis, pneumonitis, and others, observed in 48% of the concurrent QD arm, 30%

of the sequential arm, and 62% of the concurrent BID arm (Curran et al. 2000). The observed increase in normal tissue toxicity in concurrent chemoradiation treatment supports the notion that while concurrent chemoradiation enhances radiation tumoricidal effects, it simultaneously enhances injuries to normal tissues. As cancer therapy continues to explore newer agents in combination with radiation, balancing the therapeutic effect and treatment-related toxicity will also continue to challenge oncologists.

11.6.2.1

Paclitaxel Chemoradiation for NSCLC

Despite the convincing evidence from randomized studies of stage III NSCLC that chemoradiation is superior to radiation alone, the optimal chemotherapeutic agent(s), dosing schedule, sequence, and timing of chemoradiation to achieve the best therapeutic gain remain unknown. Because of lack of effective therapy

for stage III NSCLC, newer agents including the 3rd- generation chemotherapeutic agents have been tested with the intent to improve the efﬁ cacy of combination chemoradiation for stage III NSCLC over cisplatin- based chemoradiation (Chen and Okunieff 2004).

Among the 3rd-generation chemotherapeutic agents, paclitaxel has been the favorite chemotherapeutic agent investigated in many phase I/II clinical studies due to its putative radiosensitizing effect. Paclitaxel, as a single agent, has demonstrated response rates of 21–24% in the metastatic setting (stage IV) of NSCLC (Murphy et al. 1993). It is theoretically the ideal ra- diosensitizer due to its cytokinetic stabilization of the spindle microtubule resulting in arresting cells in the G2/M phase of the cell cycle, which is the most radiosensitive phase of the cell cycle (Sinclair and Morton 1966; Schiff and Horwitz 1980; Parness and Horwitz 1981; Manfredi et al. 1982; Kumar 1981; Rowinsky et al. 1988). The cytotoxic effect is attributed to tumor apoptosis after treatment with pacli- taxel (Milas et al. 1995, Milross et al. 1996; Jordan et al. 1996; Pukkinen et al. 1996). For the interaction with radiation treatment, in vitro studies have also demonstrated the radiosensitizing effects of paclitaxel in a variety of cancer cell lines, including cervical cancer, ovarian cancer, astrocytoma, melanoma, pancreatic carcinoma, breast cancer, colon cancer, lung cancer, prostate adenocarcinoma, and others (Tishler et al.

1992; Steren et al. 1993; Geard et al. 1993; Rodriguez et al. 1995; Lokeshwar et al. 1995; Elomaa et al. 1995;

Choy et al. 1993; Jordan et al. 1996; Pukkinen et al.

1996; Hornback et al. 1994). In addition to cell cycle and apoptotic effects, paclitaxel offers antineoplastic potential through other mechanisms such as enhancing tumor reoxygenation, thereby reducing the resistance of hypoxic cells to radiation and chemotherapy (Milas et al. 1995a). Furthermore, paclitaxel exhibits antiangiogenic effects on tumor vasculature by causing apoptosis of endothelial cells as well (Grant et al.

2003). In the clinical setting, there have been several different dose schedules of paclitaxel-based chemoradiation regimens reported for treating stage III NSCLC (Choy et al. 1994; Lau et al. 1997; Chen et al. 2003;

Rathmann et al. 1999; Kirkbride et al. 1999; Brodin et al. 2000; Havemann et al. 1995; Rosenthal et al.

2000), but the optimal dosing schedule in integrating paclitaxel with radiation remains to be deﬁ ned.

Because paclitaxel is a cell-cycle-speciﬁ c agent, its effect can theoretically be optimized by timing radiation to allow for radiation injury at the G2/M phase of cancer cells. Because normal tissue and cancer cells have different growth kinetics, minimizing normal tissue injury is also possible if information from pre-

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clinical research and pharmacokinetics are taken into consideration in the design of a clinical study. Here a phase I/II clinical study will be presented to illustrate the application of preclinical research on lung cancer cell lines and the pharmacokinetic characteristics of paclitaxel infusions to the design of the clinical study.

The hypothesis is that maximum tumor control and minimal normal tissue injury can be achieved by strategically optimizing the timing of daily irradiation and low-dose paclitaxel treatment.

11.6.2.2

Pharmacokinetic Consideration of Paclitaxel for Radiosensitization

Understanding the pharmacokinetics of paclitaxel is essential for optimizing the timing of paclitaxel chemotherapy and radiation treatments to enhance radiation effects and reduce normal tissue side effects. The pharmacokinetics of paclitaxel infusion are unique in that not only does the drug concentration infl uence the pharmacokinetics of the drug, but also the infusion time signifi cantly infl uences the pharmacokinetics of plasma concentration. Furthermore, bone marrow toxicity is directly related to the infusion time and drug concentration. Pharmacokinetic studies have reported a nonlinear disposition of paclitaxel in humans. In the reported clinical trials, the infusion time of paclitaxel has varied signifi cantly:

1 h, 3 h, 24 h, or continuous. Gianni et al. (1995) com-

pared the nonlinear disposition of paclitaxel with different drug concentrations and infusion times.

As demonstrated in Fig. 11.6.1a, the shorter infusion time of 3 h resulted in a fairly rapid increase to a high peak plasma level at 2–3 h after infusion, which was followed by a rapid decline of plasma concentrations.

The longer infusion time of 24 h was associated with a rise in plasma level to a moderately high plateau level for 24 h, followed by a slower decline of the drug concentrations.

As described by the same investigators, the decrease of absolute neutrophil counts from paclitaxel treatment was directly associated with the “duration” of plasma drug concentration above 0.05 µM (Fig. 11.6.1b). Thus, the long plateau of plasma concentration ≥0.05 µM from a 24-h infusion was associated with more myelotoxicity, and the shorter infusion time of 3 h was associated with less myelotoxicity despite the higher initial peak plasma level.

In the design of a clinical study when neutropenia is a concern and when radiosensitization is the primary interest, the choice of the shorter infusion time will be the logical one.

11.6.2.3

Paclitaxel Cell Cycle Eﬀ ect on Human Cancer Cell Lines

The cell cycle effect of paclitaxel on human cancer cell lines was investigated in cell lines A431, A549, and

Fig. 11.6.1 a Plasma paclitaxel concentration vs. time profi les of representative patients who received the drug at various indi- cated doses and infusion durations. Symbols represent actual measured plasma paclitaxel concentrations and lines represent model fi t curves. b Pharmacokinetic/pharmacodynamic relationship between duration spent at a plasma paclitaxel concentra- tion ≥0.05µM/l and percentage reduction in absolute neutrophil count in the fi rst course of therapy. Symbols represent individu- als treated at different doses and schedules. Curve depicts the sigmoid E_max model fi t to the data. The broken portion of the curve represents that region for which data were not available. Adapted from Gianni et al. (1995). Reprinted with permission from the American Society of Clinical Oncology

a b

Time (h) Time plasma [PACLITAXEL] ≥ 0.05 µM (h)

Plasma [PACLITAXEL] (µM) % Decrease in ANC

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NCI-H520. A431 is a human epidermoid cancer cell line; A549 and NCI-H520 are both human lung cancer cell lines. Fig. 11.6.2a demonstrates the cell cycle progression of cell line A431 after a 3 h treatment with 1µM paclitaxel. G2/M accumulation was ap- preciated at approximately 4 h after drug treatment.

Further cell cycle progression to the G2/M phase was observed and was followed by a dynamic redistribu- tion of cell cycle to return to the baseline by 48 h.

In an attempt to sustain the G2/M cell cycle arrest for maximal radiosensitizing effect, an in vitro study was conducted by pulsing paclitaxel every 48 h and analyzing the cell cycle effect 24 h later. Fig. 11.6.2b shows that by pulsing paclitaxel on alternate days using one-third of the concentration (0.33 µM), there was sustained cell cycle arrest in the G2/M phase for 6 days. Sustained G2/M cell cycle arrest by pulsing paclitaxel at lower doses was observed in all three cancer cell lines and supported the application of this schedule to the design of a clinical trial (Chen et al. 2003a).

11.6.2.4

Paclitaxel Apoptotic Eﬀ ect

In the current study, cells were treated with paclitaxel for 3 h and assayed by the Tunnel assay for apoptosis. As shown in Fig. 11.6.3, although the percent-

age of apoptotic cells varied among these three cell lines, common observations were made in that higher doses (600 nM and 2 µM) of paclitaxel caused more apoptosis than the lowest dose (200 nM). It was also observed that more apoptosis occurred at 48 h after drug treatment than at 24 h. The timing of maximal paclitaxel apoptotic effect also supports pulsing paclitaxel treatment every 48 h (Chen et al. 2003a).

11.6.2.5

Schedule-Dependent Radiosensitizing Eﬀ ect In Vitro

In preliminary studies, Keng et al. (2000) and Chen et al. (2001) investigated the schedule dependence of paclitaxel interaction with radiation in three lung cancer cell lines. Cells were treated in the culture with either 50 nM or 100 nM of paclitaxel for 3 h. The control received no paclitaxel. Radiation at various doses was delivered either immediately after the 3-h drug treatment (labeled as 3 h) or 21 h after drug treatment (labeled as 24 h). The clono- genic survival was assayed 2 weeks after radiation.

The survival curves showed that delaying radiation resulted in fewer survival clones. When other cell lines were tested, it appeared that paclitaxel chemopotentiation of radiation was often through the sub-additive mechanism rather than an addi-

Fig. 11.6.2 a Human epidermoid cell line A431 culture was exposed to 1.0 µM paclitaxel for 3 h.

After 3 h the drug-containing medium was removed and replaced with fresh culture medium.

At different time intervals, cells were analyzed for cell cycle progression. Data show that treatment with paclitaxel caused G2/M arrest at approximately 4 h posttreatment, maximizing at 24 h. This was followed by a timely reversal of G2/M arrest to the baseline level by 48 h. b Cells were treated with paclitaxel for 3 h using pulsed dose schedules: Drug treatment with 0.33 µM on day 1 (B3); or day 1 and day 3 (B4);

or day 1, day 3, and day 5 (B5). Drug-containing culture medium was removed after treatment and replaced with fresh maintenance medium.

The cells were analyzed for cell cycle distribution at 24 h after drug removal. B1 shows the cell cycle distribution of baseline without drug treatment. B2 shows the maximal arrest of G2/

M 24 hours after treatment with 1.0 µM paclitaxel. B3–B5 show that treatment with pulsed paclitaxel three times a week using one-third of the initial dose sustained the G2/M cell cycle effect. Adapted from Chen et al. (2003a), and used with permission

a

b

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tive or synergistic mechanism. Even in the subad- ditive situation, delaying radiation resulted in fewer surviving clones than immediate irradiation after drug treatment.

11.6.2.6

Clinical Protocol of Pulsed Paclitaxel and Radiation

A phase I/II clinical study for stage III NSCLC was designed taking into account the pharmacokinetic information, the timing of radiosensitization, and the cell cycle and apoptotic effect of paclitaxel from the preclinical studies. A 1 h infusion time was chosen to allow for a short exposure time to the drug for sensitization, followed by a rapid clearing of plasma drug concentration to avoid sensitizing normal tissues and to allow for less cytotoxicity to bone marrow. The clinical trial design dictated low-dose paclitaxel infusion in the morning on Monday, Wednesday, and Friday. Thoracic radiation (XRT) was given after 4:00 PM on the days when patients received paclitaxel, to allow for a minimum of a 4 h interval for cell cycle progression. On Tuesday and Thursday, when there was no paclitaxel treatment, XRT was given any time after 11:00 AM. For the phase I study, the starting dose of paclitaxel was 15 mg/m² with a dose es-

calation in 5 mg/m²increments. The average XRT dose was 60–65 Gy to gross disease and 45–58 Gy to microscopic disease, given at 1.8 Gy daily fractions over 6–7.5 weeks. Forty-one patients were enrolled (23 for the phase I study and 18 for the phase II study), and 33 completed treatment. Eight patients did not complete protocol treatments due to acute allergic reactions in three, distant disease spread during therapy in three, and two intercur- rent deaths unrelated to therapy. Stage distribution of patients with NSCLC was two in stage I, one in stage IIB, 15 in stage IIIA, and 22 in stage IIIB. One patient had stage II mesothelioma.

11.6.2.7

Tumor Response, In-Field Tumor Control, and Survival

Almost all tumors had a remarkable and durable response to therapy using this regimen. Table 11.6.1 demonstrates the tumor response in the phase I study.

Mean tumor shrinkage was 82%±14%, 84%±16%, and 84%±27% for dose levels I, II, and III, respec- tively, with an average primary tumor shrinkage at 4–6 weeks posttherapy of 83%±7% (95% conﬁ dence interval). The overall locoregional tumor response rate in the phase I study was 100% (2/17 [12%] complete response and 15/17 [88%] partial response).

Fig. 11.6.4 demonstrates an example of radiographic tumor shrinkage 4 weeks after treatment in a patient with stage III NSCLC. Local control for those patients who completed radiotherapy in both phase I and II was 98%, with a median follow-up of 11 months. For patients who did not complete the 7.5-week protocol treatment, the survival was dismal. For those who completed the protocol treatment, the survival estimate was 52% at 1 year, 40% at 2 years, and 21%

at 3 years, and survival for all patients enrolled was 46% at 1 year, 33% at 2 years, and 18 % at 3 years (Table 11.6.2). The in-ﬁ eld local control was durable up to the last day of follow-up for most patients, which has been demonstrated by the PET scans ob- tained 3 years later in some patients (Chen et al.

2003a).

11.6.2.8 Toxicity

As anticipated, low-dose pulsed paclitaxel chemoradiation was associated with low toxicity. There was no treatment interruption from side effects of

Fig. 11.6.3 Human cancer cell lines A431, A549, and NCI-H520 were treated with paclitaxel at 200 nM, 600 nM, or 2 µM concentrations. Cells were analyzed for apoptosis using Tunnel assay at 24 h as well as 48 h after drug treatment. Data showed that apoptosis was more prominent at a higher drug concentration and was observed primarily at 48 h after drug treatment.

Adapted from Chen et al. (2003a), and used with permission

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therapy. Toxicity was assessed for all patients in the phase I study, including one patient who received only two-thirds of the total radiation dose.

There were no patients who experienced grade 3 or 4 neutropenia, thrombocytopenia, neuropathy, or cardiac arrhythmia. Three of 18 patients (17%) experienced grade 3 pneumonitis, and 3/18 (17%) experienced grade 3 esophagitis. No patients experienced grade 4 pneumonitis or esophagitis (Chen et al. 2001).

11.6.2.9

Future Directions in Chemopotentiation of Radiotherapy for Stage III NSCLC

At least eight different dosing schedules for paclitaxel chemoradiation have been employed in clinical trials for treating stage III NSCLC; as a whole, they

Table 11.6.1 Tumor response 4–6 weeks after pulsed low-dose paclitaxel chemoradiation. Adapted from Chen et al. (2003a) with permission. (CR complete response [disappearance of tumor], PR partial response [>50% volume reduction])

Pulsed paclitaxel Tumor Response rate dose level shrinkage

CR PR

I, 15 mg/m² (n=6) 82%±14% 2 4 II, 20 mg/m² (n=6) 84%±16% 0 6 III, 25 mg/m² (n=4) 84%±27% 0 4

Average 83%±7% (95% CI) 14% 86%

Fig. 11.6.4 Tumor response by CT scans. A large lesion of non-small cell lung cancer presented with superior vena cava compression in the right upper lobe of the lung. The lesion had completely disappeared 4 weeks after pulsed paclitaxel chemoradiation. Adapted from Chen et al. (2003), and used with permission

Table 11.6.2 Survival and local tumor control comparisons for stage III NSCLC treated with combination chemoradiation. Adapted from Chen et al. (2003a) with permission (RT radiotherapy)

Chemoradiation trials 2-year 3-year Local

survival survival control

Chen et al.: Pulsed Taxol and RT

All patients 33% 18% N/A

Patients completing therapy 40% 21% 98%

Schaake-Koning et al. (EORTC)

Chemoradiation arm 26% 16% 30%^a

RT arm 13% --- 19%

Furuse et al. (Japanese)

Concurrent chemo arm 35% 22% --- Sequential chemo arm 27% 15% ---

Dillman et al. (CALGB)

Chemoradiation arm 26% 23% ---

RT arm 13% 11% ---

Arriagada et al. (French)

Chemoradiation arm 21% 11% 17%^b

RT arm 14% 5% 15%

Sause et al. (RTOG8808)

Chemoradiation arm 32% 17% ---

RT arm 22% 11% ---

Morton et al. (NCCT)

Chemoradiation arm 21% --- 46%^c

RT arm 16% --- 45%

a Actuarial local control at 2 years

b Local control at 1 year

c Local control at time of ﬁ rst relapse

pre-treatment

1 month post-treatment

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targeted therapy, gene therapy, hypoxic cell-targeted therapy, or other innovative therapeutic approaches may help prevent distant metastasis.

11.6.3

Cytokine Markers for Radiation Pulmonary Injury

11.6.3.1

Radiation Lung Injury

Due to normal tissue constraints, high-dose radiation to the tumoricidal dose for chest tumors has not been feasible using standard external beam radiation therapy. For standard radiation treatment to the chest, radiation dose has been limited to 60–65 Gy in order to avoid serious pneumonitis, esophageal stricture, ﬁ stula, or cardiac damage. Indeed, normal tissue injury from combined modality treatment for lung cancer has been the major dose-limiting factor for aggressive chemoradiation treatment. Through clinical experience using chemoradiation combined modality therapy for lung cancer, higher rates of both esophagitis and pneumonitis have been consistently observed (Chen and Okunieff 2004). Although interstitial pneumonitis has been recognized as a distinct clinical complication of cancer therapy, no routine diagnostic testing has been established as a simple way to assess the risk of pneumonitis prior to cancer treatment. Although most interstitial pneumonitis from cancer therapy is self-limiting, serious and potentially lethal incidences have been observed following all treatment modalities, including radiation therapy (Yorke et al. 2002; Jenkins et al.

2003; Ash-Bernal et al. 2002; Reckzeh et al. 1996), chemotherapy (Thomas et al. 2000; Sleijfer 2001;

Wang et al. 2001), and even some of the more recently developed molecularly-targeted therapies (Rosado et al. 2003; Burton et al. 2003).

Among all types of interstitial pneumonitis caused by different agents, radiation-induced pneumonitis has been the most widely investigated, both in clinical and in laboratory research. The peak incidence of radiation pneumonitis is between 6 weeks and 3 months after completion of radiation treatments.

However, it can also occur unexpectedly, with little or no warning; therefore, many attempts have been made to identify clinical risk factors for its onset. Clinical studies have reported a number of clinical factors, including total radiation dose, irradiated lung volume exceeding 20 Gy, mean lung dose, fractionation, have shown both mixed response rates and toxic-

ity proﬁ les. These schedules include concurrent radiation with continuous infusion of paclitaxel, daily paclitaxel, twice-weekly paclitaxel, thrice-weekly paclitaxel, weekly paclitaxel, and once-every-3–4-weeks paclitaxel as a single agent or in combination with a second chemotherapeutic agent. The overall response rates have been in the range of 65–100%, but gener- ally treatment toxicities also have been high, particu- larly in neutropenia, thrombocytopenia, esophagitis, and pneumonitis (Chen and Okunieff 2004). These effects are attributed to the radiosensitizing effect of paclitaxel on normal tissues as well.

Stage III NSCLC often presents with large tumor volume for the primary and the regional nodal disease. If the primary and regional tumors are not con- trolled, distant metastasis cannot be prevented. The lack of effective local therapy for inoperable NSCLC has been well recognized. Biopsy of tumors after therapy for locally advanced NSCLC resulted in only 17%

local tumor control after chemoradiation therapy and only 15% after radiotherapy alone (Arriagada et al. 1991). Although chemoradiation is the current standard of care for stage III NSCLC, improvements in local disease control and survival have been modest, despite aggressive clinical investigations using numerous chemotherapeutic agents in conjunction with radiation in many phase I/II studies (Chen and Okunieff 2004). Among these dose schedules, the pulsed paclitaxel chemoradiation schedule, which was based on the preclinical research model, yielded superior primary tumor response and ultimate local control. These results support the idea that preclinical studies using cancer cell lines or animal models to address the sequence and timing of treatment modalities may prove more fruitful in gaining favor- able clinical outcomes than those regimens built on an empirical basis. The superior local tumor control produced by pulsed paclitaxel chemoradiation for stage III NSCLC has provided new information in the understanding of cancer biology for this tumor stage. This regimen has overcome the local failure of stage III NSCLC for primary chest tumors as large as 10–12 cm at the time of presentation. The differential between the signiﬁ cant gain of local disease control (98% by pulsed paclitaxel vs. historical control of large randomized studies of 46% at best) vs. the modest survival gain (3-year survival of 21% vs. 15% of large studies of historical control) emphasizes that future therapy should aim not only for chemopotentiation for chest disease control, but also for more effective prevention and treatment of distant micrometastasis (Table 11.6.2). Antiangiogenic therapy, molecularly

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daily fraction size, performance status, pretreatment pulmonary function, gender, low pretreatment blood oxygen, high C-reactive protein, and others (Roach et al. 1995; Kwa et al. 1998; Graham et al. 1999;

Inoue et al. 2001; Segawa et al. 1997; Robnett et al.

2000; Hernando et al. 2001). However, despite the identiﬁ cation of these clinical contributing factors, clinical research has not led to the development of a reliable and simple diagnostic laboratory test that could predict the risk of postradiation pneumonitis and, in particular, one that could be administered before starting therapy. The development of such a test using patient blood specimens or lavage ﬂ uid from bronchial washing is highly desirable because of the unpredictable nature of serious adverse events, which occur sporadically and without reliable clinical warning signs; its use prior to or during the early phase of therapy would allow clinicians to customize and modify therapy intensity for those patients at higher risk for serious interstitial pneumonitis.

11.6.3.2

Cytokine and Radiation Lung Injury

Progress made in recent years has increased our understanding of the cellular and molecular mechanisms involved in radiation lung injury. We have come to appreciate the complexity of radiation pneumonitis, now seen as a process involving active interaction among resident cells of the lung parenchyma and circulatory immune and infl ammatory cells. There is increasing evidence of immune cells augmenting pneumonitis through complex autocrine, paracrine, and systemic regulatory mechanisms critically or- chestrated by cytokines. Early studies suggested that radiation-induced lung injury was characterized by alveolar infi ltrates of mononuclear cells, primarily CD4⁺ T cells and macrophages/monocytes (mononuclear alveolitis), while exhibiting a relative lack of neutrophils (Fryer et al. 1978; Roberts et al. 1993), a common marker for infectious processes. In addition, several studies using bronchoscopy have confi rmed this fi nding in patients with active pneumonitis, dem- onstrating the presence of mononuclear cells without signifi cant numbers of neutrophils in the lavage fl uids (Maasilta et al. 1993; Nakayama et al. 1996).

Ultimately, mononuclear alveolitis is followed by progressive fi brosis of the lung as well as the deposition and accumulation of collagen fi bers and extracellular matrix. Clinically, a decline in lung volume, compli- ance, and diffusion capacity is an unavoidable long- term consequence of lung fi brosis.

Animal research has induced multiple humoral factors (cytokines) in the lung by ionizing radiation (Rubin et al. 1995; Johnston et al. 1998; Hallahan et al. 1997, 2002). Rubin et al. (1995) reported a cascade of cytokine induction including interleukin 1α (IL-1α), transforming growth factor ß1 (TGF-ß1), and TGF-ß2 in lung tissue after radiation in animal research models. Because most animal research has been conducted in large single fractions of radiation, the relevance to cancer patients receiving fractionated daily low-dose radiation remains unclear. However, based on data from animal research models, it is hy- pothesized that when normal lung tissue is exposed to chemotherapy, radiation, oxygen, tumor necrosis factor (TNF-α), or other foreign insult such as lipo- polysaccharide, a cascade of cellular and humoral events occurs as a tissue response to injury (Chen et al. 2003). There is interaction among alveolar epi- thelium (type I and type II pneumocytes), vascular endothelium, fi broblasts, lymphocytes, and macrophages through the humoral factors such as adhesion molecules, chemokines, infl ammatory cytokines (TNF-α, IL-6, and IL-1), and fi brotic cytokines (basic fi broblast growth factor [bFGF], TGF-ß, and plate- let-derived growth factor [PDGF]). Chemokines and adhesion molecules mediate leukocyte traffi cking, extravasation from the vascular compartment, and homing to sites of infl ammation (Kaseda et al. 2000;

Butcher et al. 1996; Hallahan et al. 1997; Ding et al. 2000). Macrophages and lymphocytes are further recruited from the bone marrow to the chest, causing alveolar inﬁ ltrates during the pneumonitic phase.

At least three different classes of cytokines have been reported to correlate with the risk of radiation pneumonitis in patients: the inﬂ ammatory cytokines IL-1α and IL-6, the proﬁ brotic cytokine TGF-β1, and intercellular adhesion molecule-1 (ICAM-1). These cytokines will be discussed in further detail.

11.6.3.3

Inﬂ ammatory Cytokines IL-1α and IL-6

Radiation pneumonitis can be regarded as an ab- errant inﬂ ammatory response to radiation injuries.

Both IL-1α and IL-6 are cytokines central to regulation of immunity and inﬂ ammatory responses. These cytokines are important immunoregulatory moieties and share some common immune functions. Both cytokines are pleiotropic inﬂ ammatory cytokines, recognized as "acute phase response" proteins. They are chemotactic for mononuclear cells, and they ac- tivate lymphocytes, regulate fevers, and precipitate a

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ﬁ brovascular response. While the source of IL-1α is primarily from monocytes and alveolar macrophages (Elias et al. 1985; O’Brien-Lander et al. 1993), IL- 6 is synthesized by a variety of cells, including the alveolar macrophages, endothelium, type II pneumocytes, T lymphocytes, and lung ﬁ broblasts (Kelley 1990; Cromwell et al. 1992; Crestani et al. 1994).

In vitro experiments have shown that alveolar macrophages exposed to radiation release increased quantities of IL-1α and IL-1β (O’Brien-Lander et al. 1993). It has also been shown that IL-1α stimu- lates human lung fi broblasts to produce IL-6 and stabilizes IL-6 messenger RNA production (Elias and Lentz 1990). Others have reported IL-1 induction of IL-6 in a variety of cells, including thymocytes, and have suggested that IL-6 is involved in many of the pleiotropic effects of IL-1 (Helle et al. 1988). Such in vitro work indeed suggests a regulatory relationship between the two infl ammatory cytokines. Chen et al. (2001a, 2002) analyzed a panel of cytokines in serial blood samples of cancer patients receiving thoracic radiation. Radiation pneumonitis was diag- nosed using the National Cancer Institute common toxicity criteria. Cytokine analysis was assayed for IL-1α, IL-6, MCP-1, E-selectin, L-selectin, P-selectin, TGF-ß1, and basic bFGF, using enzyme linked immu- nosorbent assay (ELISA). Among all these cytokines analyzed, only the two infl ammatory cytokines, IL-6 and IL-1α, were persistently higher in patients who subsequently developed pneumonitis after radiation.

Additionally, patients with high pretreatment circulating IL-6 and IL-1α levels had a greater chance of developing subsequent pneumonitis after therapy (Fig. 11.6.5), suggesting the possibility of using pretreatment IL-1α and IL-6 levels to predict patients at risk for radiation pneumonitis.

11.6.3.4

Chemokines and Cell Adhesion Molecules

Tissue inﬂ ammation requires leukocytes to undergo transendothelial migration and extravasation of the vascular compartment into sites of inﬂ ammation.

Chemokines and adhesion molecules are key cytokines in facilitating leukocyte recruitment to the site of inﬂ ammation (Kaseda et al. 2000; Butcher et al. 1996; Hallahan et al. 1997; Ding et al. 2000).

Johnston et al. (1998) investigated a panel of chemokines including monocyte chemotactic protein (MCP)-1, lymphotactin (Ltn), RANTES, eotaxin, macrophage inﬂ ammatory protein (MIP-1α, -1ß, and -2), and interferon-inducible protein 10 (IP-10)

in fi brosis-prone mice (C57BL/6) and fi brosis-resistant mice (C3H/HeJ). In these studies, local MCP-1 and IP-10 expressions did not increase 8 weeks after lung radiation, but a signifi cantly higher level of expression was found only in the fi brosis-prone mice 26 weeks after lung radiation. Hallahan et al.

(1997) investigated the role of adhesion molecules in animal lungs after radiation. They found an increase of ICAM-1 and E-selectin expression in vascular endothelium after radiation. In the animal model, they found that anti-ICAM-1 blocking antibody at- tenuated the infl ammatory cell infi ltration to the lungs of mice after radiation exposure. In addition, using ICAM-1 knockout mice, which were defi cient in expressing ICAM-1 after radiation, radiation-induced pulmonary infl ammatory cell infi ltration was abrogated (Hallahan et al. 1997a, 2002). Research in chemokine and adhesion molecules in cancer patients produced some correlated fi ndings. Ishii and Kitamura (1999) reported that patients with an increase of ICAM-1 in circulating cytokine levels during radiation had a higher risk of radiation

Fig. 11.6.5 a Circulating IL-1α (in pg/ml) before radiation, weekly during radiation, and after completing radiation treat- ments. b Circulating IL-6 (in pg/ml) before radiation, weekly during radiation, and after completing radiation treatments.

Adapted from Chen et al. (2001a) and from Chen et al. (2002);

used with permission from both publishers

b a

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pneumonitis. Chen et al. (2002) investigated the role of other chemokine and adhesion molecules in the blood of cancer patients, including MCP-1, E-selectin, L-selectin, and P-selectin, but they found no correlation with the risk of radiation lung injury.

11.6.3.5

Fibrotic Cytokine: Transforming Growth Factor β TGF-ß is a key modulator in the induction of fi brosis, alterations in collagen metabolism, and increase in extracellular matrix formation following radiation therapy (Burger et al. 1998; Barcelloss-Hoff 1998; Randall and Coggle 1995; Rodemann and Bamberg 1995). TGF-ß has been shown to be a growth factor regulating fi broblast proliferation, differentiation, and matrix production under physi- ological as well as pathophysiological conditions (Kovacs 1991; Fine and Goldstein 1987; Sigel et al. 1996; Zhang and Jacobberger 1996). It was found to be a primary cytokine for radiation-induced fi brosis (Rodemann and Bamberg 1995). In animal research models, different investigators have found that TGF-ß expression is induced after lung radiation and is the major mediator for postradiation lung fi brosis (Finkelstein et al. 1994; Rubin et al. 1992;

Franko et al. 1997). The search for TGF-ß as a marker for radiation lung injury in patients has been com- plicated by the fact that lung cancers may produce TGF-β. Nonetheless, Anscher et al. (1998) found a correlation between higher risk of radiation lung injury in patients with high circulating levels of TGF-β near the end of radiotherapy. This observation was signiﬁ cant for the patients with normal pretreatment TGF-β levels but not signiﬁ cant for those with higher than normal pretreatment TGF-β levels.

11.6.4 Conclusions

For decades, radiobiologists and radiation oncologists have searched for ways to enhance radiation effects on human solid tumors. Fletcher (1973) reported the clinical dose-response curves of human malignant epithelial tumors based on clinical expe- riences at M.D. Anderson Cancer Center in Houston.

In this report, the volume effect on tumor control probability was explored, and the concept of larger tumors requiring higher radiation doses to effec- tively control the tumor by radiation was introduced.

Oncologists at the University of Florida also reported a similar ﬁ nding for squamous cell carcinoma of the head and neck region. The dose required to eradicate solid tumors is proportional to the tumor volume:

60–65 Gy for tumors smaller than 1.0 cm in diam- eter, 65–70 Gy for tumors 1.5–2.0 cm, 70–75 Gy for tumors 2.5–3.0 cm, and 75–80 Gy for tumors 3.5–6.0 cm (Million et al. 1994). If one extrapolates Fletcher’s estimate, it would require an 80–100 Gy radiation dose to eradicate large epithelial tumors of stage III NSCLC. Such a high dose to the chest has not been possible in the clinical setting due to potential fatal pneumonitis. Clinical practice in treating stage III NSCLC in general has kept radiation doses below 65 Gy and has therefore produced unsatisfactory local regional disease control for patients in this stage.

If gross tumors in the chest cannot be eradicated, distant failures are deﬁ nite sequelae.

Thus, optimization of chemotherapy and radiation continues to deserve preclinical studies aimed at maximizing the primary tumor control while keeping the toxicity low. For enhancing local tumor control, more frequent but lower doses of drug appear par- ticularly promising in maximizing tumor response and minimizing toxicity. An interesting observation was made in this phase I study of pulsed paclitaxel chemoradiation: The lack of dose response with increasing paclitaxel doses beyond 15 mg/m² supports that when low-dose chemotherapy is used as a radiation-sensitizing agent, escalating chemotherapy doses may not further potentiate radiation but rather may increase the toxicity of therapy. The concept of a

“minimally effective dose” for radiation potentiation by chemotherapy is contradictory to the traditional strategy of systemic chemotherapy when achieving a maximally tolerated dose is the primary goal. The superior tumor response and the durable tumor control achieved by pulsed low dose paclitaxel chemoradiation are intriguing. This dramatic local tumor effect cannot be explained by cell cycle and apoptotic effects of pulsed paclitaxel alone. More recently, small and frequent dosing of chemotherapeutic agents has been shown to suppress tumor growth through the mechanism of antiangiogenesis in animal tumor models (Boehm et al. 1997; Browder et al. 2000).

Other potential mechanisms that may contribute to the effectiveness of the small frequent dosing schedule of paclitaxel and radiation should be further investigated in animals.

Recent discoveries in translational research using specimens from lung cancer patients have allowed a major breakthrough in our understanding of the so- matic mutation of epidermal growth factor receptor

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(EGFR) in relation to tumor response to treatments by geﬁ tinib (Iressa). Clinically, geﬁ tinib offers an overall 10% response rate for patients with NSCLC.

Examinations of the EGFR genes have revealed that mutations at the tyrosine kinase domain not only spe- ciﬁ cally predicted responders to geﬁ tinib treatments but also showed a striking correlation with patient characteristics: Mutations were more frequent in ad- enocarcinomas than in other NSCLC, more frequent in women than in men, and more frequent in patients from Japan than in those from the U.S. (Lynch et al.

2004; Paez et al. 2004). Serious lung injury was infre- quently observed in large studies of patients treated with geﬁ tinib for NSCLC, and included rare but fatal interstitial pneumonitis occurring with higher incidence in the Japanese patients (1.87%) than in patients outside Japan (0.35%).

Such ﬁ ndings support endeavors in identifying gene and molecular markers, not only for the efﬁ - cacy of therapy but also for identifying markers for normal tissue injury. Simultaneous measurement of normal tissue consequences in translational research is critical, as the ultimate goal of treatment must include minimizing the acute and long-term toxicity to normal tissues. Cancer therapy has evolved to- wards a multiagent and multitarget approach, with the inclusion of gene therapy, antiangiogenic therapy, inhibition of epidermal growth factor pathways, inhibition of oncogene pathways, inhibition of other signal transduction pathways, hypoxic cell target- ing therapy, and others (Chen et al. 2003). Balancing therapeutic effects versus normal tissue effects will become more complex and more challenging to oncologists in the future.

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