Biology Contribution
Preclinical Evaluation of Dose-Volume Effects and Lung Toxicity Occurring In and Out-of-Field

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Purpose

The aim of this study was to define the dose and dose-volume relationship of radiation-induced pulmonary toxicities occurring in and out-of-field in mouse models of early inflammatory and late fibrotic response.

Materials and Methods

Early radiation-induced inflammation and fibrosis were investigated in C3H/NeJ and C57BL/6J mice, respectively. Animals were irradiated with 20 Gy delivered to the upper region of the right lung as a single fraction or as 3 consecutive fractions using the Small Animal Radiation Research Platform (Xstrahl Inc, Camberley, UK). Cone beam computed tomography was performed for image guidance before irradiation and to monitor late toxicity. Histologic sections were examined for neutrophil and macrophage infiltration as markers of early inflammatory response and type I collagen staining as a marker of late-occurring fibrosis. Correlation was evaluated with the dose-volume histogram parameters calculated for individual mice and changes in the observed cone beam computed tomography values.

Results

Mean lung dose and the volume receiving over 10 Gy (V10) showed significant correlation with late responses for single and fractionated exposures in directly targeted volumes. Responses observed outside the target volume were attributed to nontargeted effects and showed no dependence on either mean lung dose or V10.

Conclusions

Quantitative assessment of normal tissue response closely correlates early and late pulmonary response with clinical parameters, demonstrating this approach as a potential tool to facilitate clinical translation of preclinical studies. Out-of-field effects were observed but did not correlate with dosimetric parameters, suggesting that nontargeted effects may have a role in driving toxicities outside the treatment field.

Introduction

Radiation-induced lung toxicity (RILT) is the most common dose-limiting adverse sequela in patients receiving thoracic irradiation.1, 2 Despite technological advances in conformal delivery techniques, dose escalation has failed to demonstrate significant overall survival benefits compared with previously established lower-dose regimens, as demonstrated by the Radiation Therapy Oncology Group trial 0671).3, 4 Furthermore, as both the number and age of cancer survivors increase, addressing critical gaps in understanding normal tissue responses is of significant importance in improving quality of life after radiation therapy.5, 6

RILT describes multiple pulmonary pathologies that can develop weeks to years after radiation therapy and significantly compromise quality of life.7 It represents a spectrum of biological events evolving from the initial stages of early inflammation, symptomatic radiation pneumonitis (RP), through late stages of radiation-induced fibrosis. Clinical scoring systems for RILT use a combination of radiologic, functional, and symptomatic criteria to determine a global toxicity score,8 yet prediction of patients most likely to develop RILT remains challenging in the clinic and may be an important factor in optimizing personalized radiation therapy.

Over the past 2 decades, progress has been made in defining normal tissue tolerances.9, 10 By establishing the dose-volume relationship between the delivered dose and resulting normal tissue injury, dose escalation can be achieved with minimum impact on normal tissue complication.8 Several dose thresholds used in clinical practice have been investigated, with strong correlation observed between different dosimetric parameters (V5 and V20) within individual institutions.10 However, these parameters are dependent on radiation therapy techniques specific to each institution, making it difficult to establish general dose-volume constraints based on retrospective clinical data.8, 10

Preclinical studies of pulmonary response have largely investigated the dose-response using whole-thorax irradiation.11, 12 Previous studies have used partial-thorax irradiation to explore the differential sensitivity of different lung structures and the relationship of radiation dose and volume to damage and morbidity in the mouse lung. These studies identified a heterogeneous response of the lung to partial-volume irradiation and suggested that this is due to critical target cells located in the base and apex of the mouse lung. Moreover, proton irradiation studies indicated a change in dose-limiting toxicity with the change of the irradiated volume.13, 14, 15 Although these studies have provided vital information on the pulmonary radiation response of different rodent strains and defined critical structures and dose thresholds for lung toxicities, the implementation of small-animal irradiators has enabled major refinements in the precision and accuracy of dose delivery, allowing more clinically relevant dose distributions and fractionation schedules to be achieved experimentally.16, 17, 18

This study aimed to characterize early and late pulmonary responses of partially irradiated lung volumes using small-animal image guided radiation therapy. Biological responses occurring in and out-of-field were quantified and compared with dosimetric parameters such as mean lung dose (MLD) and V10 to determine their dose-volume dependence. This study supports the development of refined preclinical models of RILT, incorporating clinical dosimetric parameters and the role of nontargeted effects occurring outside the treatment field.

Section snippets

Preclinical study design

RILT that manifested as early inflammation (RP) and late toxicity (radiation-induced fibrosis) was investigated within and outside of the irradiated target volume in C3H/NeJ and C57BL/6J mice, respectively. All animals were obtained from Charles River Laboratories (Oxford, UK) and irradiated at 8 to 10 weeks old under UK Department of Health–approved procedures. A minimum of 5 mice per experimental group were sacrificed at each of the time points investigated, along with time-matched

In-field effects

Early inflammatory responses were determined as the level of neutrophil and macrophage infiltration in the target volume after irradiation, as shown in Figures 2a and 2b. In CBCT-only control animals, cell counts of 115 ± 6.27 cells/mm2 were observed for neutrophils and 97 ± 5.05 cells/mm2 for macrophages. At 72 hours after irradiation with 20 Gy, the numbers of inflammatory cells increased to 312.3 ± 36.79 cells/mm2 for neutrophils and to 220.6 ± 12.31 cells/mm2 for macrophages (P < .0002),

Discussion

This study aimed, first, to demonstrate correlations between early and late pulmonary toxicities with clinical dose and volume parameters after partial-lung irradiation and, second, to characterize these effects occurring within the target volume and out-of-field. Many clinical studies have focused on analyzing specific DVH parameters to assess the risk of RILT development.5, 8, 21, 22 These studies report that the percentage of the total lung volume exceeding 20 Gy (V20) and the MLD are

Conclusions

Specific cellular responses in preclinical models can be quantitatively assessed in the context of clinically relevant parameters. Significant correlations were observed between the MLD and V10 for late pulmonary toxicity in field. Toxicities were observed outside the targeted volume but showed no dependence on MLD and V10, suggesting that nontargeted radiation effects may have a role in driving toxicities outside the treatment field.

Acknowledgments

This work was supported by Cancer Research UK Grant Number A20842. MG would like to acknowledge Tom Simms Memorial Fund and Queen's Foundation, Queen's University Belfast. SOO is supported by HSC Research and Development Division, in Public Health Agency Northern Ireland.

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    Conflict of interest: none.

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