Generation and Differentiation of Adult Tissue-Derived Human Thyroid Organoids.

Total thyroidectomy as part of thyroid cancer treatment results in hypothyroidism requiring lifelong daily thyroid hormone replacement. Unbalanced hormone levels result in persistent complaints such as fatigue, constipation, and weight increase. Therefore, we aimed to investigate a patient-derived thyroid organoid model with the potential to regenerate the thyroid gland. Murine and human thyroid-derived cells were cultured as organoids capable of self-renewal and which expressed proliferation and putative stem cell and thyroid characteristics, without a change in the expression of thyroid tumor-related genes. These organoids formed thyroid-tissue-resembling structures in culture. (Xeno-)transplantation of 600,000 dispersed organoid cells underneath the kidney capsule of a hypothyroid mouse model resulted in the generation of hormone-producing thyroid-resembling follicles. This study provides evidence that thyroid-lineage-specific cells can form organoids that are able to self-renew and differentiate into functional thyroid tissue. Subsequent (xeno-)transplantation of these thyroid organoids demonstrates a proof of principle for functional miniature gland formation.

Addition of HER2 and CD44 to 18F-FDG PET-based clinico-radiomic models enhances prediction of neoadjuvant chemoradiotherapy response in esophageal cancer.

OBJECTIVES: To assess the complementary value of human epidermal growth factor receptor 2 (HER2)-related biological tumor markers to clinico-radiomic models in predicting complete response to neoadjuvant chemoradiotherapy (NCRT) in esophageal cancer patients. METHODS: Expression of HER2 was assessed by immunohistochemistry in pre-treatment tumor biopsies of 96 patients with locally advanced esophageal cancer. Five other potentially active HER2-related biological tumor markers in esophageal cancer were examined in a sub-analysis on 43 patients. Patients received at least four of the five cycles of chemotherapy and full radiotherapy regimen followed by esophagectomy. Three reference clinico-radiomic models based on 18F-FDG PET were constructed to predict pathologic response, which was categorized into complete versus incomplete (Mandard tumor regression grade 1 vs. 2-5). The complementary value of the biological tumor markers was evaluated by internal validation through bootstrapping. RESULTS: Pathologic examination revealed 21 (22%) complete and 75 (78%) incomplete responders. HER2 and cluster of differentiation 44 (CD44), analyzed in the sub-analysis, were univariably associated with pathologic response. Incorporation of HER2 and CD44 into the reference models improved the overall performance (R2s of 0.221, 0.270, and 0.225) and discrimination AUCs of 0.759, 0.857, and 0.816. All models exhibited moderate to good calibration. The remaining studied biological tumor markers did not yield model improvement. CONCLUSIONS: Incorporation of HER2 and CD44 into clinico-radiomic prediction models improved NCRT response prediction in esophageal cancer. These biological tumor markers are promising in initial response evaluation. KEY POINTS: • A multimodality approach, integrating independent genomic and radiomic information, is promising to improve prediction of γpCR in patients with esophageal cancer. • HER2 and CD44 are potential biological tumor markers in the initial work-up of patients with esophageal cancer. • Prediction models combining 18F-FDG PET radiomic features with HER2 and CD44 may be useful in the decision to omit surgery after neoadjuvant chemoradiotherapy in patients with esophageal cancer.

Hedgehog Pathway as a Potential Intervention Target in Esophageal Cancer.

Esophageal cancer (EC) is an aggressive disease with a poor prognosis. Treatment resistance is a major challenge in successful anti-cancer therapy. Pathological complete response after neoadjuvant chemoradiation (nCRT) is low, thus requiring therapy optimization. The Hedgehog (HH) pathway has been implicated in therapy resistance, as well as in cancer stemness. This article focusses on the HH pathway as a putative target in the treatment of EC. Immunohistochemistry on HH members was applied to EC patient material followed by modulation of 3D-EC cell cultures, fluorescence-activated cell sorting (FACS), and gene expression analysis after HH pathway modulation. Sonic Hedgehog (SHH) and its receptor Patched1 (PTCH1) were significantly enriched in EC resection material of patients with microresidual disease (mRD) after receiving nCRT, compared to the control group. Stimulation with SHH resulted in an up-regulation of cancer stemness in EC sphere cultures, as indicated by increased sphere formation after sorting for CD44+/CD24- EC cancer stem-like cell (CSC) population. On the contrary, inhibiting this pathway with vismodegib led to a decrease in cancer stemness and both radiation and carboplatin resistance. Our results strengthen the role of the HH pathway in chemoradiotherapy resistance. These findings suggest that targeting the HH pathway could be an attractive approach to control CSCs.

Decreasing Irradiated Rat Lung Volume Changes Dose-Limiting Toxicity From Early to Late Effects.

PURPOSE: Technological developments in radiation therapy result in smaller irradiated volumes of normal tissue. Because the risk of radiation therapy-induced toxicity generally depends on irradiated volume, changing volume could change the dose-limiting toxicity of a treatment. Recently, in our rat model, we found that early radiation-induced lung dysfunction (RILD) was closely related to irradiated volume dependent vascular remodeling besides inflammation. The exact relationship between early and late RILD is still unknown. Therefore, in this preclinical study we investigated the dose-volume relationship of late RILD, assessed its dependence on early and late pathologies and studied if decreasing irradiated volume changed the dose-limiting toxicity. METHODS AND MATERIALS: A volume of 25%, 32%, 50%, 63%, 88%, or 100% of the rat lung was irradiated using protons. Until 26 weeks after irradiation, respiratory rates were measured. Macrovascular remodeling, pulmonary inflammation, and fibrosis were assessed at 26 weeks after irradiation. For all endpoints dose-volume response curves were made. These results were compared to our previously published early lung effects. RESULTS: Early vascular remodeling and inflammation correlated significantly with early RILD. Late RILD correlated with inflammation and fibrosis, but not with vascular remodeling. In contrast to the early effects, late vascular remodeling, inflammation and fibrosis showed a primarily dose but not volume dependence. Comparison of respiratory rate increases early and late after irradiation for the different dose-distributions indicated that with decreasing irradiated volumes, the dose-limiting toxicity changed from early to late RILD. CONCLUSIONS: In our rat model, different pathologies underlie early and late RILD with different dose-volume dependencies. Consequently, the dose-limiting toxicity changed from early to late dysfunction when the irradiated volume was reduced. In patients, early and late RILD are also due to different pathologies. As such, new radiation techniques reducing irradiated volume might change the dose-limiting toxicity of the radiation therapy treatment.

Sparing the region of the salivary gland containing stem cells preserves saliva production after radiotherapy for head and neck cancer.

Each year, 500,000 patients are treated with radiotherapy for head and neck cancer, resulting in relatively high survival rates. However, in 40% of patients, quality of life is severely compromised because of radiation-induced impairment of salivary gland function and consequent xerostomia (dry mouth). New radiation treatment technologies enable sparing of parts of the salivary glands. We have determined the parts of the major salivary gland, the parotid gland, that need to be spared to ensure that the gland continues to produce saliva after irradiation treatment. In mice, rats, and humans, we showed that stem and progenitor cells reside in the region of the parotid gland containing the major ducts. We demonstrated in rats that inclusion of the ducts in the radiation field led to loss of regenerative capacity, resulting in long-term gland dysfunction with reduced saliva production. Then we showed in a cohort of patients with head and neck cancer that the radiation dose to the region of the salivary gland containing the stem/progenitor cells predicted the function of the salivary glands one year after radiotherapy. Finally, we showed that this region of the salivary gland could be spared during radiotherapy, thus reducing the risk of post-radiotherapy xerostomia.

ACE inhibition attenuates radiation-induced cardiopulmonary damage.

BACKGROUND AND PURPOSE: In thoracic irradiation, the maximum radiation dose is restricted by the risk of radiation-induced cardiopulmonary damage and dysfunction limiting tumor control. We showed that radiation-induced sub-clinical cardiac damage and lung damage in rats mutually interact and that combined irradiation intensifies cardiopulmonary toxicity. Unfortunately, current clinical practice does not include preventative measures to attenuate radiation-induced lung or cardiac toxicity. Here, we investigate the effects of the ACE inhibitor captopril on radiation-induced cardiopulmonary damage. MATERIAL AND METHODS: After local irradiation of rat heart and/or lungs captopril was administered orally. Cardiopulmonary performance was assessed using biweekly breathing rate measurements. At 8 weeks post-irradiation, cardiac hemodynamics were measured, CT scans and histopathology were analyzed. RESULTS: Captopril significantly improved breathing rate and cardiopulmonary density/structure, but only when the heart was included in the radiation field. Consistently, captopril reduced radiation-induced pleural and pericardial effusion and cardiac fibrosis, resulting in an improved left ventricular end-diastolic pressure only in the heart-irradiated groups. CONCLUSION: Captopril improves cardiopulmonary morphology and function by reducing acute cardiac damage, a risk factor in the development of radiation-induced cardiopulmonary toxicity. ACE inhibition should be evaluated as a strategy to reduce cardiopulmonary complications induced by radiotherapy to the thoracic area.

Purification and ex vivo expansion of fully functional salivary gland stem cells.

Hyposalivation often leads to irreversible and untreatable xerostomia. Salivary gland (SG) stem cell therapy is an attractive putative option to salvage these patients but is impeded by the limited availability of adult human tissue. Here, using murine SG cells, we demonstrate single-cell self-renewal, differentiation, enrichment of SG stem cells, and robust in vitro expansion. Dependent on stem cell marker expression, SG sphere-derived single cells could be differentiated in vitro into distinct lobular or ductal/lobular organoids, suggestive of progenitor or stem cell potency. Expanded cells were able to form miniglands/organoids containing multiple SG cell lineages. Expansion of these multipotent cells through serial passaging resulted in selection of a cell population, homogenous for stem cell marker expression (CD24(hi)/CD29(hi)). Cells highly expressing CD24 and CD29 could be prospectively isolated and were able to efficiently restore radiation-damaged SG function. Our approach will facilitate the use of adult SG stem cells for a variety of scientific and therapeutic purposes.

Salisphere derived c-Kit+ cell transplantation restores tissue homeostasis in irradiated salivary gland.

INTRODUCTION: During radiotherapy salivary glands of head and neck cancer patients are unavoidably co-irradiated, potentially resulting in life-long impairment. Recently we showed that transplantation of salisphere-derived c-Kit expressing cells can functionally regenerate irradiated salivary glands. This study aims to select a more potent subpopulation of c-Kit(+) cells, co-expressing stem cell markers and to investigate whether long-term tissue homeostasis is restored after stem cell transplantation. METHODS AND RESULTS: Salisphere derived c-Kit(+) cells that co-expressed CD24 and/or CD49f markers, were intra-glandularly injected into 15 Gy irradiated submandibular glands of mice. Particularly, c-Kit(+)/CD24(+)/CD49f(+) cell transplanted mice improved saliva production (54.59 ± 11.1%) versus the irradiated control group (21.5 ± 8.7%). Increase in expression of cells with differentiated duct cell markers like, cytokeratins (CK8, 18, 7 and 14) indicated functional recovery of this compartment. Moreover, ductal stem cell marker expression like c-Kit, CD133, CD24 and CD49f reappeared after transplantation indicating long-term functional maintenance potential of the gland. Furthermore, a normalization of vascularization as indicated by CD31 expression and reduction of fibrosis was observed, indicative of normalization of the microenvironment. CONCLUSIONS: Our results show that stem cell transplantation not only rescues hypo-salivation, but also restores tissue homeostasis of the irradiated gland, necessary for long-term maintenance of adult tissue.

Prediction of response to radiotherapy in the treatment of esophageal cancer using stem cell markers.

BACKGROUND AND PURPOSE: In this study, we investigated whether cancer stem cell marker expressing cells can be identified that predict for the response of esophageal cancer (EC) to CRT. MATERIALS AND METHODS: EC cell-lines OE-33 and OE-21 were used to assess in vitro, stem cell activity, proliferative capacity and radiation response. Xenograft tumors were generated using NOD/SCID mice to assess in vivo proliferative capacity and tumor hypoxia. Archival and fresh EC biopsy tissue was used to confirm our in vitro and in vivo results. RESULTS: We showed that the CD44+/CD24- subpopulation of EC cells exerts a higher proliferation rate and sphere forming potential and is more radioresistant in vitro, when compared to unselected or CD44+/CD24+ cells. Moreover, CD44+/CD24- cells formed xenograft tumors faster and were often located in hypoxic tumor areas. In a study of archival pre-neoadjuvant CRT biopsy material from EC adenocarcinoma patients (N=27), this population could only be identified in 50% (9/18) of reduced-responders to neoadjuvant CRT, but never (0/9) in the complete responders (P=0.009). CONCLUSION: These results warrant further investigation into the possible clinical benefit of CD44+/CD24- as a predictive marker in EC patients for the response to chemoradiation.

Physiological interaction of heart and lung in thoracic irradiation.

INTRODUCTION: The risk of early radiation-induced lung toxicity (RILT) limits the dose and efficacy of radiation therapy of thoracic tumors. In addition to lung dose, coirradiation of the heart is a known risk factor in the development RILT. The aim of this study was to identify the underlying physiology of the interaction between lung and heart in thoracic irradiation. METHODS AND MATERIALS: Rat hearts, lungs, or both were irradiated to 20 Gy using high-precision proton beams. Cardiopulmonary performance was assessed using breathing rate measurements and F(18)-fluorodeoxyglucose positron emission tomography ((18)F-FDG-PET) scans biweekly and left- and right-sided cardiac hemodynamic measurements and histopathology analysis at 8 weeks postirradiation. RESULTS: Two to 12 weeks after heart irradiation, a pronounced defect in the uptake of (18)F-FDG in the left ventricle (LV) was observed. At 8 weeks postirradiation, this coincided with LV perivascular fibrosis, an increase in LV end-diastolic pressure, and pulmonary edema in the shielded lungs. Lung irradiation alone not only increased pulmonary artery pressure and perivascular edema but also induced an increased LV relaxation time. Combined irradiation of lung and heart induced pronounced increases in LV end-diastolic pressure and relaxation time, in addition to an increase in right ventricle end-diastolic pressure, indicative of biventricular diastolic dysfunction. Moreover, enhanced pulmonary edema, inflammation and fibrosis were also observed. CONCLUSIONS: Both lung and heart irradiation cause cardiac and pulmonary toxicity via different mechanisms. Thus, when combined, the loss of cardiopulmonary performance is intensified further, explaining the deleterious effects of heart and lung coirradiation. Our findings show for the first time the physiological mechanism underlying the development of a multiorgan complication, RILT. Reduction of dose to either of these organs offers new opportunities to improve radiation therapy treatment of thoracic tumors, potentially facilitating increased treatment doses and tumor control.

Volume-dependent expression of in-field and out-of-field effects in the proton-irradiated rat lung.

PURPOSE: To investigate whether occurrence of early radiation effects in lung tissue depends on local dose only. METHODS AND MATERIALS: Twenty-five percent, 50%, 66%, 88%, or 100% of the rat lung was irradiated using single fractions of 150-MeV protons. For all volumes, in-field and out-of-field dose-response curves were obtained 8 weeks after irradiation. The pathohistology of parenchymal inflammation, infiltrates, fibrosis, and vascular damage and the relative expression of proinflammatory cytokines interleukin (IL)-1α, transforming growth factor-ß, IL-6, and tumor necrosis factor-α were assessed. RESULTS: For all histologic endpoints, irradiated dose- and volume-dependent in-field and out-of-field effects were observed, albeit with different dynamics. Of note, the out-of-field effects for vascular damage were very similar to the in-field effects. Interestingly, only IL-6 showed a clear dose-dependent increase in expression both in-field and out-of-field, whereas the expression levels of IL-1α, transforming growth factor-ß, and tumor necrosis factor-α were either very low or without a clear dose-volume relation. As such, none of the radiation effects studied depended only on local dose to the tissue. CONCLUSION: The effects of radiation to lung tissue do not only depend on local dose to that tissue. Especially at high-volume irradiation, lung damage seems to present globally rather than locally. The accuracy of predictive modeling may be improved by including nonlocal effects.

Quantifying local radiation-induced lung damage from computed tomography.

PURPOSE: Optimal implementation of new radiotherapy techniques requires accurate predictive models for normal tissue complications. Since clinically used dose distributions are nonuniform, local tissue damage needs to be measured and related to local tissue dose. In lung, radiation-induced damage results in density changes that have been measured by computed tomography (CT) imaging noninvasively, but not yet on a localized scale. Therefore, the aim of the present study was to develop a method for quantification of local radiation-induced lung tissue damage using CT. METHODS AND MATERIALS: CT images of the thorax were made 8 and 26 weeks after irradiation of 100%, 75%, 50%, and 25% lung volume of rats. Local lung tissue structure (S(L)) was quantified from local mean and local standard deviation of the CT density in Hounsfield units in 1-mm(3) subvolumes. The relation of changes in S(L) (DeltaS(L)) to histologic changes and breathing rate was investigated. Feasibility for clinical application was tested by applying the method to CT images of a patient with non-small-cell lung carcinoma and investigating the local dose-effect relationship of DeltaS(L). RESULTS: In rats, a clear dose-response relationship of DeltaS(L) was observed at different time points after radiation. Furthermore, DeltaS(L) correlated strongly to histologic endpoints (infiltrates and inflammatory cells) and breathing rate. In the patient, progressive local dose-dependent increases in DeltaS(L) were observed. CONCLUSION: We developed a method to quantify local radiation-induced tissue damage in the lung using CT. This method can be used in the development of more accurate predictive models for normal tissue complications.

Bath and shower effects in the rat parotid gland explain increased relative risk of parotid gland dysfunction after intensity-modulated radiotherapy.

PURPOSE: To assess in a rat model whether adding a subtolerance dose in a region adjacent to a high-dose irradiated subvolume of the parotid gland influences its response (bath-and-shower effect). METHODS AND MATERIALS: Irradiation of the whole, cranial 50%, and/or the caudal 50% of the parotid glands of Wistar rats was performed using 150-MeV protons. To determine suitable (i.e., subtolerance) dose levels for a bath-dose, both whole parotid glands were irradiated with 5 to 25 Gy. Subsequently groups of Wistar rats received 30 Gy to the caudal 50% (shower) and 0 to 10 Gy to the cranial 50% (bath) of both parotid glands. Stimulated saliva flow rate (function) was measured before and up to 240 days after irradiation. RESULTS: Irradiation of both glands up to a dose of 10 Gy did not result in late loss of function and is thus regarded subtolerance. Addition of a dose bath of 1 to 10 Gy to a high-dose in the caudal 50% of the glands resulted in enhanced function loss. CONCLUSION: Similar to the spinal cord, the parotid gland demonstrates a bath and shower effect, which may explain the less-than-expected sparing of function after IMRT.

Enhanced proliferation of acinar and progenitor cells by prophylactic pilocarpine treatment underlies the observed amelioration of radiation injury to parotid glands.

BACKGROUND: Administration of pilocarpine before irradiation can ameliorate radiation-induced hyposalivation. Indirect evidence suggests that this effect may be mediated through induction of a compensatory response. In this study, this hypothesis is tested directly, by assessing the proliferation of progenitor and secretory cells in irradiated and non-irradiated parotid gland tissue. METHODS: In a rat model, parotid glands were unilaterally irradiated with a single dose of 15 Gy, 60 min after administration of pilocarpine (4.0mg/kg). Rats were sacrificed for proliferating cell nuclear antigen (PCNA) labelling, assessing the number of proliferating progenitor and secretory cells, before, and 10h, 1, 3, 7, 10, 20 and 30 days after irradiation. RESULTS: A small radiation-induced increase in PCNA expressing cells was observed, both in the acinar (secretory cells) and intercalated duct cell (containing the progenitor cells) compartment. This increment was significantly enhanced in pilocarpine pre-treated glands. In fact, in this group of animals increased proliferation was observed both in the irradiated and the shielded gland. CONCLUSIONS: Amelioration of early loss of rat salivary gland function after radiation by pilocarpine pre-treatment is, at least in part, due to compensatory mechanisms through increased proliferation of undamaged cells.

Rescue of salivary gland function after stem cell transplantation in irradiated glands.

Head and neck cancer is the fifth most common malignancy and accounts for 3% of all new cancer cases each year. Despite relatively high survival rates, the quality of life of these patients is severely compromised because of radiation-induced impairment of salivary gland function and consequential xerostomia (dry mouth syndrome). In this study, a clinically applicable method for the restoration of radiation-impaired salivary gland function using salivary gland stem cell transplantation was developed. Salivary gland cells were isolated from murine submandibular glands and cultured in vitro as salispheres, which contained cells expressing the stem cell markers Sca-1, c-Kit and Musashi-1. In vitro, the cells differentiated into salivary gland duct cells and mucin and amylase producing acinar cells. Stem cell enrichment was performed by flow cytrometric selection using c-Kit as a marker. In vitro, the cells differentiated into amylase producing acinar cells. In vivo, intra-glandular transplantation of a small number of c-Kit(+) cells resulted in long-term restoration of salivary gland morphology and function. Moreover, donor-derived stem cells could be isolated from primary recipients, cultured as secondary spheres and after re-transplantation ameliorate radiation damage. Our approach is the first proof for the potential use of stem cell transplantation to functionally rescue salivary gland deficiency.

Optimum dose range for the amelioration of long term radiation-induced hyposalivation using prophylactic pilocarpine treatment.

BACKGROUND: To determine dose and time dependency of pilocarpine pre-treatment protection from late damage after unilateral irradiation of the rat parotid gland. METHODS AND MATERIALS: The right parotid gland of saline (1mg/ml) or pilocarpine (4 mg/kg) pre-treated rats was irradiated with 10, 15 and 20 Gy. Saliva was collected from the irradiated and shielded parotid before, 30, 60, 120 and 240 days after irradiation. The number of acinar cells/gland was determined 30, 120 and 240 days after irradiation by histological examination. RESULTS: Pilocarpine pre-treated rats, protection of parotid gland function was seen in the early-intermediate phase (0-120 days) after 15 Gy and in the late phase (>120 days) after 10 and 15 Gy. Although no protection was observed after 20 Gy, a stimulatory effect of pilocarpine on the non-irradiated gland resulted in a significant increase in total saliva secretion. The increase in function after pilocarpine treatment was paralleled by a significant increase in the number of acinar cells in both the irradiated and shielded glands. CONCLUSIONS: Pre-irradiation treatment with pilocarpine induces compensatory response, at lower doses, in the irradiated and at higher doses in the non-irradiated gland reducing late damage, due to stimulation of unirradiated or surviving cells to divide.

The impact of heart irradiation on dose-volume effects in the rat lung.

PURPOSE: To test the hypothesis that heart irradiation increases the risk of a symptomatic radiation-induced loss of lung function (SRILF) and that this can be well-described as a modulation of the functional reserve of the lung. METHODS AND MATERIALS: Rats were irradiated with 150-MeV protons. Dose-response curves were obtained for a significant increase in breathing frequency after irradiation of 100%, 75%, 50%, or 25% of the total lung volume, either including or excluding the heart from the irradiation field. A significant increase in the mean respiratory rate after 6-12 weeks compared with 0-4 weeks was defined as SRILF, based on biweekly measurements of the respiratory rate. The critical volume (CV) model was used to describe the risk of SRILF. Fits were done using a maximum likelihood method. Consistency between model and data was tested using a previously developed goodness-of-fit test. RESULTS: The CV model could be fitted consistently to the data for lung irradiation only. However, this fitted model failed to predict the data that also included heart irradiation. Even refitting the model to all data resulted in a significant difference between model and data. These results imply that, although the CV model describes the risk of SRILF when the heart is spared, the model needs to be modified to account for the impact of dose to the heart on the risk of SRILF. Finally, a modified CV model is described that is consistent to all data. CONCLUSIONS: The detrimental effect of dose to the heart on the incidence of SRILF can be described by a dose dependent decrease in functional reserve of the lung.

Relation between radiation-induced whole lung functional loss and regional structural changes in partial irradiated rat lung.

PURPOSE: Radiation-induced pulmonary toxicity is characterized by dose, region, and time-dependent severe changes in lung morphology and function. This study sought to determine the relation between the structural and functional changes in the irradiated rat lung at three different phases after irradiation. MATERIALS AND METHODS: Six groups of animals were irradiated to 16-22 Gy to six different lung regions, each containing 50% of the total lung volume. Before and every 2 weeks after irradiation, the breathing rate (BR) was measured, and at Weeks 8, 26, and 38 CT was performed. From the computed tomography scans, the irradiated lung tissue was delineated using a computerized algorithm. A single quantitative measure for structural change was derived from changes of the mean and standard deviation of the density within the delineated lung. Subsequently, this was correlated with the BR in the corresponding phase. RESULTS: In the mediastinal and apex region, the BR and computed tomography density changes did not correlate in any phase. After lateral irradiation, the density changes always correlated with the BR; however, in all other regions, the density changes only correlated significantly (r(2) = 0.46-0.85, p < 0.05) with the BR in Week 26. CONCLUSION: Changes in pulmonary function correlated with the structural changes in the absence of confounding heart irradiation.

Secondary radiation damage as the main cause for unexpected volume effects: a histopathologic study of the parotid gland.

PURPOSE: To elucidate with a histopathological study the mechanism of region-dependent volume effects in the partly irradiated parotid gland of the rat. METHODS AND MATERIALS: Wistar rats were locally X-irradiated with collimators with conformal radiation portals for 100% volume and 50% cranial/caudal partial volumes. Single doses up to 40 Gy were applied. Parotid saliva samples were collected, and the three lobes of the parotid gland were examined individually on the macro- and micromorphologic level up to 1 year after irradiation. RESULTS: Dose-dependent loss of gland weight was observed 1 year after total or partial X-irradiation. Weight loss of the glands correlated very well with loss of secretory function. Irradiating the cranial 50% volume (implicating a shielded lateral lobe) resulted in substantially more damage in terms of weight loss and loss of secretory function than 50% caudal irradiation (shielding the ventral and dorsal lobe). Histologic examinations of the glands 1 year after irradiation revealed that the shielded lateral lobe was severely affected, in contrast to the shielded ventral and dorsal lobes. Time studies showed that irradiation of the cranial 50% volume caused late development of secondary damage in the shielded lateral lobe, becoming manifest between 240 and 360 days after irradiation. The possible clinical significance of this finding is discussed. CONCLUSION: It is concluded that the observed region-dependent volume effect for late function loss in the rat parotid gland after partial irradiation is mainly caused by secondary events in the shielded lateral lobe. The most probable first step (primary radiation event) in the development of this secondary damage is radiation exposure to the hilus region (located between the ventral and dorsal lobe). By injuring major excretory ducts and supply routes for blood and nerves in this area, the facility system necessary for proper functioning of the nonexposed lateral lobe is seriously affected. The unexpected volume effect in the rat might have consequences for treatment strategies in radiotherapy, implicating not only salivary glands but also other organs with a seemingly homogeneous distribution of radiosensitive elements, a situation wherein volume effects have not been anticipated up to now.

Radioprotective effect of amifostine on parotid gland functioning is region dependent.

PURPOSE: To investigation the protective ability of amifostine during partial irradiation of the rat parotid gland. METHODS AND MATERIALS: Single-dose X-ray irradiation was performed by use of collimators with conformal radiation portals for either the 100% volume (15 Gy) or the 50% cranial/caudal partial parotid gland volumes (30 Gy). Amifostine was administered intraperitoneally at a dose of 250 mg per kg body weight, 25 minutes before irradiation. Saliva flow rates, gland weights, and the tissues of the individual lobes were investigated up to 1 year after treatment. RESULTS: A clear protective effect of amifostine was found against loss of saliva flow, the altered appearance of gross morphology, loss of gland weight, and histopathologic changes for the 100% volume gland irradiations and for the 50% volume cranial irradiations but not for the 50% volume caudal irradiations. CONCLUSIONS: The protective ability of amifostine is strongly dependent on the irradiated glandular region and observed for later damage only. The major effect of the drug seems to be the prevention of volume effects caused by secondary damage that occurs in shielded parts of the gland. The results of the present study show that understanding of the anatomy and physiology of organs that are to be spared is necessary to ensure optimal preservation of function.