2.2 Radiation Therapy

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2.2 Radiation Therapy

2.2.1 Radiobiology of Normal Lung Tissue and Lung Tumours

Yuta Shibamoto and Masaki Hara

Y. Shibamoto, MD, DMSc, Professor and Chairman,

Department of Radiology, Nagoya City University Graduate School of Medical Sciences, 1 Kawasumi, Mizuho-cho, Mizuho- ku, Nagoya, 467-8601, Japan

M. Hara, MD, PhD, Associate Professor,

Department of Radiology, Nagoya City University Graduate School of Medical Sciences, 1 Kawasumi, Mizuho-cho, Mizuho- ku, Nagoya, 467-8601, Japan

CONTENTS

2.2.1.1 Introduction 59

2.2.1.2 Biology of Irradiated Normal Lung Tissues 59 2.2.1.3 Radiosensitivity and Proliferative Activity Testing

of Primary Lung Cancers 60

2.2.1.4 18F-Fluorodeoxyglucose Positron Emission Tomography Findings of Primary Lung Cancers 63 2.2.1.5 RadGenomics Project 64

References 64

2.2.1.1 Introduction

Radiation biology has a history dating back almost 100 years. It began soon after Roentgen discovered X rays in 1895. In the early 1900s, Bergonie and Tribondeau formulated the famous rule that radiosensitivity of cells or tissues is correlated with the frequency of mitoses which they undergo and that poorly-differentiated and fast-proliferating tissues are more radiosensitive than well-differentiated and slow-proliferating tissues, from experiments using the testis of rats. Since then, biological studies have been carried out mostly using animals. In the 1950s, Puck and Marcus (1956) devised a colony formation method with which survival of cells became measur- able in vitro. This facilitated tremendous progress in radiation biology at the cellular level. Most of the important biological phenomena at the cellular and animal levels appeared to have been clariﬁ ed by 1980.

More recently, advances in molecular biology have brought us much new knowledge on radiobiology

of various normal tissues and tumours. Although molecular mechanisms for normal tissue reaction and tumour cell killing are not completely clariﬁ ed yet, many cytokines have been identiﬁ ed that are involved in the pathogenesis of normal tissue reaction including radiation pneumonitis. Some of the more recent research efforts aim at identifying single nucleotide polymorphism of genes and oncogene expression that are possibly related to increased normal tissue reaction and radiosensitivity of tumours. In this article, we introduce recent investigations including our own on radiobiology of normal lung tissue and lung tumours.

2.2.1.2

Biology of Irradiated Normal Lung Tissues

Previously, clonogenic death of target cells had been considered a major cause of normal tissue injury by irradiation. Regarding lung injury caused by irradiation, for example, the type II pneumocyte has been considered one of the most important target cells. The type II pneumocyte exhibited the earli- est response to radiation and a decrease in lamel- lar bodies and a corresponding increase in alveo- lar surfactant were reported shortly after radiation (Penny et al. 1982). By 18–63 weeks, several type II cells underwent degeneration and sloughing into al- veolar spaces. This would remain true, but recent re- searches have revealed that inﬂ ammatory cytokines are also involved in the pathogenesis of radiation pneumonitis. Rubin et al. (1995) demonstrated early and persistent elevation of cytokine production and gene expression following pulmonary irradiation in mice. Among these cytokines and genes are interleu- kin-1α/β, transforming growth factor-beta (TGF-β), platelet-derived growth factor, and collagen and ﬁ - bronectin genes. The expression started shortly after irradiation and continued cascade-like for several months. They advocated that there is no latent period in a biologic sense for development of delayed

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radiation pneumonitis. Anscher et al. (1994) reported that plasma TGF-β1 levels measured during radiation therapy for lung cancer may be useful in identifying patients who will or will not go on to develop symptomatic radiation pneumonitis. A sub- sequent study by the group suggested that changes in plasma TGF-β1 levels during radiotherapy may be a useful means by which to identify patients at risk for the development of symptomatic radiation pneumonitis, particularly in the subset of patients whose pre-treatment TGF-β levels are over 7.5 ng/ml (Anscher et al. 1997).

Chen et al. (2002) measured changes in plasma levels of IL-1α, IL-6, monocyte chemotactic protein 1, E-selectin, L-selectin, TGF-β1, and basic fi broblast growth factor in patients undergoing thoracic radiotherapy. They found that both IL-1α and IL-6 levels were signifi cantly higher before, during, and after radiotherapy for those who had radiation pneumonitis. None of the other cytokines appeared to defi - nitely correlate with radiation pneumonitis. Their results are different from those reported by other in- vestigators (Rubin et al. 1995; Anscher et al. 1997), and further investigations with a larger number of patients are encouraged to determine cytokines most closely related to the development of radiation pneumonitis.

As a therapeutic approach, Rabbani et al. (2003) investigated soluble TGF-β type II receptor gene therapy to ameliorate acute pulmonary radiation injury in rats. They administered recombinant human adenoviral vector carrying this gene before 30 Gy of hemithoracic irradiation with an expectation that it might reduce availability of active TGF-β1 and thereby protect the lung. They observed a signifi cant increase in the plasma level of TGF-β1 type II receptor after injection of the vector, and a signifi cant reduction in respiratory rates at 4 weeks after treatment. These fi ndings were supported by histological analyses of the irradiated lung tissue. Their study showed the ability of gene therapy to induce an increase in circu- lating levels of soluble receptors, to reduce the tissue level of active TGF-β1, and consequently to ameliorate acute radiation-induced lung injury.

Molecular aspects of radiation injury of the lung, including the role of respective cytokines and genes, are still to be clariﬁ ed. Fruitful application of the molecular biology to therapeutic approaches may be expected in future. In the pathogenesis of radiation injury of normal tissues other than the lung, various cytokines have been reported to be involved (Chiang et al. 1997; Richter et al. 1997). Also, radiation-induced cellular senescence may be related to

the development of late radiation morbidity (Peters 1996). In the near future, it is hoped that the relationship between clonogenic death of target cells, cytokine induction, and radiation-induced senescence is clariﬁ ed at the molecular level.

2.2.1.3

Radiosensitivity and Proliferative Activity Testing of Primary Lung Cancers

The necessity for tailor-made cancer treatment based on the biological characteristics of each tumour has been advocated recently. For this purpose, prediction of tumour and/or normal tissue sensitivity to treatment is necessary. To estimate radiosensitivity of tumour cells, several types of predictive assays have been proposed. Among them, the SF-2 assay in which the surviving fraction of tumour cells at 2 Gy of in vitro irradiation is measured using colony formation or colorimetric methods has been most intensively investigated. The reliability of the SF-2 assay to predict radiosensitivity has remained rather controversial, but West et al. (1997), who made the greatest contribution to establishment of the SF-2 assay, reported a clear correlation between the SF-2 and the prognosis after radiation therapy in uterine cervical cancers; patients with tumours showing SF- 2 values below the median had a better prognosis than those with tumours showing SF-2 values above the median. However, the use of the SF-2 assay for radiosensitivity prediction has not become a com- monly used tool in clinics. One of the reasons for this may be the long waiting time before obtaining assay results.

We have tried to establish a more rapid assay of radiosensitivity using the cytokinesis-block micronucleus (MN) test. MN formation represents chro- mosomal damage and the MN frequency increases with radiation dose. Using this assay, we devised a method of simultaneously estimating radiosensitivity and proliferative activity of human tumours (Shibamoto et al. 1994, 1998). Estimation of tumour proliferative activity is also important in radiotherapy, since rapid growing tumours are considered to be resistant to protracted conventional radiotherapy.

One of the important parameters of proliferative activity is the potential doubling time (Tpot). The Tpot, which is a doubling time in the absence of cell loss, is considered to represent repopulation rates during and after radiotherapy better than the volume doubling time. The assay involves determination of MN

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Lung SCC

Duration of culture (days)

2 4 6 8 0 2 4 4

Dose (Gy)

Nuclei/cell% MNC

100

50

2.0

1.5

1.0

0.5

MN/BNC

Fig. 2.2.1.1. Assay of a squamous cell carcinoma of the lung.

Left, the percentage of multinucleate cells (MNC) and the aver- age number of nuclei per cell as a function of culture duration.

In the latter, an exponential curve was ﬁ tted to the three points obtained on days 1–3. Right, the average number of micro- nuclei (MN) per binucleate cell (BNC) as a function of the radiation dose

frequency after radiation, the fraction of tumour cells undergoing mitosis in vitro (the dividing fraction, DF), and the extrapolated time for tumour cell nuclei to double in culture (in vitro Tpot).

After establishing the method in xenografted human and murine tumours (Shibamoto and Streffer 1991; Shibamoto et al. 1991), we have used this assay for primary human tumours. All the specimens were obtained at operation and not by biopsy.

A total of 133 specimens of various human tumours were obtained from patients receiving no preopera- tive radiation or chemotherapy. The average weight of tumour specimens obtained was 2.0 g. The tumour tissues were minced with scissors and treated at 37ºC for 2 h with 1 mg/ml collagenase/dispase solution.

After fi ltering and centrifuging the tumour cell sus- pension, the cells were plated onto multiple collagen- coated dishes (20 cm2). Whenever the cell yield was suffi cient, 3–6×105 cells per dish were plated onto ten dishes. The culture medium used was Ham F12 supplemented with 20% foetal bovine serum and 0.2 mg/ml gentamicin sulphate. Within 1 h after plat- ing, 2 or 4 Gy of irradiation was given to some of the dishes (usually two dishes per dose). Then, cytochalasin B dissolved in dimethyl sulfoxide was added to all dishes at the concentration of 1.5 µg/ml. This concentration of cytochalasin B appeared to be optimal in all of the human tumour cells tested in the previ- ous study (Shibamoto et al. 1994). Cultures were ter- minated at various intervals, and the cells were fi xed with 1% glutaraldehyde in phosphate buffer, treated with 5 N hydrochloric acid for 20 min and stained in the dark with Schiff ’s reagent for 1 h. Usually, unirradiated cells were fi xed on days 1, 2, 3, 4, 6, and 8. By monitoring the increase in the number of binucleate cells (BNC) in the unirradiated dishes, the optimal days for fi xing the irradiated cells were determined;

these were usually days 4–6.

Tumour cells were distinguished from normal cells on the basis of morphological criteria such as nuclear irregularity, dense nuclear staining and a high nucleo- cytoplasmic ratio, and only those judged as tumour cells were scored. The cells with different numbers of nuclei (mononucleate, binucleate, trinucleate, etc.) and the micronuclei in the BNC were counted under a microscope at a magniﬁ cation of 1000. At least 100 (250–300 whenever possible) cells were assessed per dish, and at least 50 (100–150 whenever possible) BNC were assessed to determine the MN frequency.

BNC with three or more micronuclei were occasion- ally found, but all micronuclei were scored. Then, the percentage of multinucleate cells (MNC, cells with two or more nuclei), the average number of nuclei

per cell, and the average number of micronuclei per single BNC (= MN frequency) were calculated. The DF (= maximal MNC percentage) and Tpot were es- timated from the unirradiated group of cultures. The Tpot obtained with this assay was the extrapolated time for the nuclear ratio (the average number of nuclei per tumour cell) to reach 2.0. When the MN frequencies at different culture times (after day 3) were not signiﬁ cantly different from each other, these values were averaged for both unirradiated and irradiated cells.

Figure 2.2.1.1 shows a representative assay result for a squamous cell carcinoma of the lung. As shown, three sets of data, i.e. DF, Tpot and MN frequency, were obtained with this assay. The proportion of MNC appeared to reach a plateau within 4–6 days in all tumour cells investigated, and the DF was de- ﬁ ned as the mean of the percent MNC at the plateau.

The Tpot was obtained by ﬁ tting the initial part (for days 1–3) of the nuclear ratio curve to an exponential curve and extrapolating from it, when necessary. This extrapolation was necessary in nearly all tumours in which the nuclear ratio did not reach 2.0. In many tumours like the squamous cell carcinoma shown in Fig. 2.2.1.1, MN frequency at 4 Gy did not increase signiﬁ cantly as compared to that at 2 Gy. This phe- nomenon has already been reported in established murine and human cell lines by Abend et al. (1995), who attributed it to the development of apoptosis at higher doses.

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Of the 133 tumours tested so far, the DF and Tpot were obtained in 104 (78%) and the MN frequency could be evaluated in 93 (70%). Table 2.2.1.1 shows the assay data for lung cancers and other tumours.

Among these tumours, metastases of pancreatic cancer had the highest DF and shortest Tpot values, while meningiomas had the lowest DF and longest Tpot.

Among malignant tumours, bladder cancer appeared to have the lowest proliferative activity. Thus, the DF and Tpot values appear to reﬂ ect the degree of ma- lignancy of each histological type of tumours. With regard to MN frequency, there was no great difference among these tumours. Especially, there was no difference in the MN frequency between adenocarcinoma and squamous cell carcinoma of the lung. This ﬁ nding agrees with the data on uterine cancer showing similar values for the surviving fraction at 2 Gy in the two types of carcinomas (West et al. 1995). Small cell lung carcinomas are more radiosensitive than non-small cell carcinomas, but in this study, although the number of small cell carcinomas was small, the MN frequency after radiation for these tumours was similar to or only slightly higher than that for non-small cell carcinomas. Small cell carcinomas appear to die more often by apoptosis (Ohmori et al. 1993), and apoptosis and MN-related death are different events (Abend et al. 1995). This may explain our observation.

shows postoperative relapse-free survival curves for the patients with Stage I–IIIB non-small cell cancer according to the DF value. The 28 patients with DF above median for each stage had signiﬁ cantly poorer relapse-free survival rate than the 28 patients with DF below the median (p=0.0077). Therefore, the DF seems to be a useful indicator of tumour proliferative activity and patient prognosis. The relationship between the DF and the growth fractions as determined by proliferation markers should be investigated, be- cause the DF values obtained in our study do not differ signiﬁ cantly from the Ki-67 or proliferating cell nuclear antigen positivity rates reported for various types of tumours.

A total of 21 patients with Stage I–IIIB non-small cell lung cancer who underwent macroscopic cura- tive surgery developed recurrence. In these patients, a correlation was found between the time to recurrence and their Tpot value (Fig. 2.2.1.3). Although we are not sure whether the regrowth rate of tumours after surgery is related to the Tpot, the Tpot obtained with this assay may also be useful in pre- dicting the postoperative period at high risk for recurrence. The ﬂ ow cytometry method is now being widely used to estimate the Tpot, but several meth- odological problems that make the obtained Tpot value inaccurate have been pointed out, including the inﬂ uence of normal cell counts in diploid tumours and interlaboratory variations (Begg 1993).

As an alternative, our method appears to be attract- ing attention recently.

Tumour n DF Tpot MN/BNC

(%) (days) (2Gy-0Gy) Lung adenocarcinoma 41 23 7.7 0.15 Lung squamous cell carcinoma 14 25 8.5 0.17 Lung large cell carcinoma 3 27 6.5 0.16 Lung small cell carcinoma 4 30 7.0 0.20 Pancreatic cancer (metastasis) 6 49 4.6 0.16 Breast cancer (metastasis) 5 27 8.5 0.16

Bladder cancer 4 15 18 0.14

Malignant glioma 4 20 9.2 0.14

Osteosarcoma (lung metastasis) 4 20 13 0.21

Meningioma 4 8.2 53 0.08

Table 2.2.1.1. Median values of the dividing fraction (DF) and potential doubling time (Tpot) and mean micronucleus frequency at 2 Gy of irradiation (after subtraction of the 0 Gy value) of various tumours

To investigate whether there is any correlation between the DF/Tpot and clinical outcome, the patients with non-small cell lung cancer at each disease stage were divided into two groups according to the DF value (above or below median of each stage). Figure 2.2.1.2

50 100

% Relapse-free

30 Month

60 DF > median (N = 28)

DF < median (N = 28) Stage I-IIIB NSCLC

Fig. 2.2.1.2. Relapse-free survival curves according to the di- viding fraction (> or < median for each stage) for patients with Stage I–IIIB non-small cell lung cancer. The stage distribution (I/II/IIIA/IIIB) was 15/4/6/3 for both groups. p=0.0077

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Only some of the patients underwent postoperative radiation therapy, but in 18 patients, including those who had cancer other than that of the lung, the clinical response of the primary or metastatic lesions could be evaluated. Figure 2.2.1.4 shows the correlation between the tumour response to radiotherapy and the MN frequency at 2 Gy of in vitro irradiation (after subtraction of the value at 0 Gy). The tumour response was classiﬁ ed as complete response (CR), partial response (PR, > 50% regression in maximal tumour area), minor response (MR, < 50 > 25% regression) or no response (NR, < 25% regression).

The MN frequency tended to be higher in tumours showing CR or PR than in those showing MR or NR.

In particular, tumours producing many micronuclei tended to show good response to radiotherapy.

However, tumours producing few micronuclei varied in their response and were not necessarily radiore- sistant. Whether the MN frequency after radiation represents the radiosensitivity is a matter of contro- versy in laboratory studies. Some authors indicated a good correlation between the MN frequency and cell survival (Shibamoto et al. 1991; Mariya et al. 1997), while others found no such correlation (Bush and McMillan 1993; Villa et al. 1994). Since cell survival is not necessarily an absolute measure of radiosensitivity, such clinical studies as ours comparing the MN frequency with actual tumour response are necessary. Since it seems possible to evaluate apoptotic cells simultaneously, it may also be worthwhile to modify the method to include scoring of both mi- cronucleated cells and apoptotic cells.

In summary, the cytokinesis-block assay is feasible in human tumour cells in primary culture. This assay provides three sets of data on the DF, Tpot, and MN frequency in approximately 1 week. The DF appears to be an index of tumour proliferative activity, and the Tpot obtained with this method was correlated with the time until recurrence. Whether or not the MN frequency after 2 Gy irradiation represents clinical radiosensitivity of the tumour is a topic of future investigation.

2.2.1.4

18F-Fluorodeoxyglucose Positron Emission Tomography Findings

of Primary Lung Cancers

Positron emission tomography (PET) using 18F-fl uo- rodeoxyglucose (FDG) is a useful tool to detect tumours with increased glucose metabolism. Although FDG uptake is observed in non-malignant lesions (Shreve et al. 1999), FDG-PET can detect small tumours of approximately 1 cm in the abdomen. We have used FDG-PET in patients with a lung nodule or suspected tumour. The majority of patients had lung cancers, but other patients with a lesion showing FDG uptake underwent surgery without preopera- tive histological confi rmation and proved to have benign diseases. We have tried to characterise FDG-PET fi ndings in these patients.

FDG-PET was performed using a GE Advance scanner (GE Medical Systems, Milwaukee, Wis., USA). The Advance unit produced 4.25-mm thick image planes (18 direct planes and 17 cross planes) with an image Potential doubling time (days)

Time to recurrence (month)

30

20

10

00 10 20

Stage I-IIIB NSCLC

Fig. 2.2.1.3. Correlation between the potential doubling time (Tpot) and time to recurrence in 21 patients with Stage I–IIIB lung cancer. R=0.70, p=0.00044

0.0 0.1 0.2 0.3 0.4

NR MR PR CR

Response to radiation

MN/BNC (2Gy – 0Gy)

Fig. 2.2.1.4. Tumour response to radiotherapy and micronu- cleus frequency at 2 Gy after subtraction of that at 0 Gy. MN/

BNC, mean number of micronuclei per binucleate cell; NR, no response; MR, minor response; PR, partial response; CR, com- plete response. Bars represent the mean for each group

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matrix of 128×128. The resolution of the scanner at full width at half maximum was 5.5 mm and the lon- gitudinal ﬁ eld view was 14.5 cm. All patients fasted for at least 6 h before imaging. A 3-min transmission scan was obtained before a 2-min emission scan by using a rotating germanium-68 pin source. Seven scans were usually performed to cover from head to thigh. Scans were started 60 min after administration of 185–170 MBq FDG.

Table 2.2.1.2 shows the standardised uptake value (SUV) of FDG and mass size in pulmonary adenocarcinoma, squamous cell carcinoma, small cell carcinoma, and tuberculoma. There was a trend whereby squamous cell carcinomas had a higher uptake than adenocarcinomas. This appeared to be partly related to the fact that adenocarcinomas are more frequently well-differentiated than squamous cell carcinomas. In adenocarcinomas, FDG uptake was higher in poorly- differentiated tumours than in well-differentiated ones, but this trend was not observed in squamous cell carcinomas. At present, we have no explanation for this discrepancy, and we will investigate further if there is any correlation between degree of histological differentiation and FDG uptake with larger numbers of patients. The SUV for small cell carcinomas were comparable to those for squamous cell carcinomas and poorly-differentiated adenocarcinomas.

Benign granulomatous diseases also showed increased FDG uptake. In our seven tuberculomas, the mean SUV was 2.5, which was comparable to that for well-differentiated and moderately-differentiated adenocarcinomas. Other benign lesions also had a moderate degree of FDG uptake, but 90% of their SUVs were below 3.0. Our results would suggest

that a lesion with an SUV of 3.0 or higher is likely to be malignant. However, an SUV below 3.0 does not necessarily indicate that the lesion is benign. It appears difﬁ cult to differentiate benign nodules from well-differentiated adenocarcinomas on the basis of SUV for FDG.

2.2.1.5

RadGenomics Project

The RadGenomics project started in April 2001 at the National Institute of Radiological Sciences in Japan (Iwakawa et al. 2002). This project promotes analysis of genes that are expressed in response to irradiation, identiﬁ cation of their allelic variants in the human population, development of an effective procedure for quantitating individual radiosensitivity, and analysis of the relationship between genetic heterogeneity and susceptibility to irradiation. Major groups of genes investigated in the project include DNA repair genes, genes for programmed cell death, genes for signal transduction, and genes for oxida- tive processes.

The outcome of the RadGenomics project should lead to improved protocols for individualised radiotherapy and reduction of adverse effects of treatment.

The project will contribute to future research on the molecular mechanisms of radiosensitivity in humans and stimulate development of new high-throughput technology for a wider application of the biological and medical sciences. Identiﬁ cation of functionally important polymorphisms in the radiation response genes may determine individual differences in sensitivity to radiation exposure or radiotherapy.

We have initiated co-operative research with the National Institute of Radiological Sciences to investigate whether there is any correlation between speciﬁ c single nucleotide polymorphism and normal tissue reaction to radiation therapy. Radiation pneumonitis is one of the major normal tissue reactions investigated in this collaboration. We will also investigate oncogene expression that is possibly related to radiosensitivity in lung cancers using a microarray.

Table 2.2.1.2. Standardised uptake ratio (SUR) of FDG in lung cancers and tuberculoma

Longest diameter(mm)

SUV

Histology n Mean SD Mean SD

Adenocarcinoma 27 26 16 2.5 1.8

Well differentiated 19 22 13 1.8 1.4 Moderately differentiated 4 34 24 3.4 1.7 Poorly differentiated 4 39 12 5.1 1.4 Squamous cell carcinoma 17 40 19 6.9 2.6 Well differentiated 3 42 12 8.1 2.7 Moderately differentiated 8 30 14 6.3 2.4 Poorly differentiated 6 51 24 7.0 2.9

Small cell carcinoma 8 40 37 5.2 2.6

Tuberculoma 7 23 10 2.5 1.3

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