Robert P. Coppes

Oral Sequelae of Head and Neck Radiotherapy

In addition to anti-tumor effects, ionizing radiation causes damage in normal tissues located in the radiation portals. Oral complications of radiotherapy in the head and neck region are the result of the deleterious effects of radiation on, e.g., salivary glands, oral mucosa, bone, dentition, masticatory musculature, and temporomandibular joints. The clinical consequences of radiotherapy include mucositis, hyposalivation, taste loss, osteoradionecrosis, radiation caries, and trismus. Mucositis and taste loss are reversible consequences that usually subside early post-irradiation, while hyposalivation is normally irreversible. Furthermore, the risk of developing radiation caries and osteoradionecrosis is a life-long threat. All these consequences form a heavy burden for the patients and have a tremendous impact on their quality of life during and after radiotherapy. In this review, the radiation-induced changes in healthy oral tissues and the resulting clinical consequences are discussed.

Prevention and treatment of the consequences of head and neck radiotherapy

The location of the primary tumor or lymph node metastases dictates the inclusion of the oral cavity, salivary glands, and jaws in the radiation treatment portals for patients who have head and neck cancer. The clinical sequelae of the radiation treatment include mucositis, hyposalivation, loss of taste, osteoradionecrosis, radiation caries, and trismus. These sequelae may be dose-limiting and have a tremendous effect on the patients quality of life. Most treatment protocols to prevent these sequelae are still based on clinical experience, but alternatives based on fundamental basic and clinical research are becoming more and more available. Many of these alternatives either need further study before they can be incorporated into the protocols commonly used to prevent and treat the radiation-related oral sequelae or await implementation of these protocols. In this review, the various possibilities for prevention and/or treatment of radiation-induced changes in healthy oral tissues and their consequences are discussed.

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.

Clinical management of salivary gland hypofunction and xerostomia in head-and-neck cancer patients: Successes and barriers

The most significant long-term complication of radiotherapy in the head-and-neck region is hyposalivation and its related complaints, particularily xerostomia. This review addresses the pathophysiology underlying irradiation damage to salivary gland tissue, the consequences of radiation injury, and issues contributing to the clinical management of salivary gland hypofunction and xerostomia. These include ways to (1) prevent or minimize radiation injury of salivary gland tissue, (2) manage radiation-induced hyposalivation and xerostomia, and (3) restore the function of salivary gland tissue damaged by radiotherapy.

Mobilization of bone marrow stem cells by granulocyte colony-stimulating factor ameliorates radiation-induced damage to salivary glands

Purpose: One of the major reasons for failure of radiotherapeutic cancer treatment is the limitation in dose that can be applied to the tumor because of coirradiation of the normal healthy tissue. Late radiation-induced damage reduces the quality of life of the patient and may even be life threatening. Replacement of the radiation-sterilized stem cells with unirradiated autologous stem cells may restore the tissue function. Here, we assessed the potential of granulocyte colony-stimulating factor (G-CSF)–mobilized bone marrow–derived cells (BMC) to regenerate and functionally restore irradiated salivary glands used as a model for normal tissue damage. Experimental Design: Male-eGFP+ bone marrow chimeric female C57BL/6 mice were treated with G-CSF, 10 to 60 days after local salivary gland irradiation. Four months after irradiation, salivary gland morphology and flow rate were assessed. Results: G-CSF treatment induced homing of large number of labeled BMCs to the submandibular glands after irradiation. These animals showed significant increased gland weight, number of acinar cells, and salivary flow rates. Donor cells expressed surface markers specific for hematopoietic or endothelial/mesenchymal cells. However, salivary gland acinar cells neither express the G-CSF receptor nor contained the GFP/Y chromosome donor cell label. Conclusions: The results show that BMCs home to damaged salivary glands after mobilization and induce repair processes, which improve function and morphology. This process does not involve transdifferentiation of BMCs to salivary gland cells. Mobilization of BMCs could become a promising modality to ameliorate radiation-induced complications after radiotherapy.

Parotid and submandibular/sublingual salivary flow during high dose radiotherapy

It was studied whether differences in acute radiosensitivity exist between parotid and submandibular/sublingual glands. The results revealed that salivary flow rates decreased dramatically during the first 2 weeks of radiotherapy. Neither recovery nor significant differences were observed between the production of saliva from the parotid and submandibular/sublingual glands during the 13 weeks observation period.

Regeneration of irradiated salivary glands with stem cell marker expressing cells

BACKGROUND Stem cell therapy could be a potential way for reducing radiation-induced hyposalivation and improving the patients quality of life. However, the identification and purification of salivary gland stem cells have not been accomplished. This study aims to better characterize the stem/progenitor cell population with regenerative potential residing in the mouse salivary gland. METHODS Mouse submandibular gland tissue, isolated cells and cultured 3 day old salispheres were tested for their expression of stem cell markers c-Kit, CD133, CD49f, and CD24 using immunohistochemistry for tissue and flow cytometry for cells. Mice were locally irradiated with a single dose of 15 Gy and transplanted with cells expressing defined markers. RESULTS Cells expressing known stem cell markers are localized in the larger ducts of the mouse salivary gland. Isolated cells and cells from day 3 salispheres also express these markers: c-Kit (0.058% vs. 0.65%), CD133 (6% vs. 5%), CD49f (78% vs. 51%), and CD24 (60% vs. 60%, respectively). Intraglandular transplantation of these cells into irradiated salivary glands of mice resulted in stem cell marker-specific recovery of salivary gland function. CONCLUSIONS Different stem cell-associated markers are expressed in mouse salivary gland cells, which upon transplantation are able to regenerate the irradiation damaged salivary gland.

Early to late sparing of radiation damage to the parotid gland by adrenergic and muscarinic receptor agonists

Damage to salivary glands after radiotherapeutic treatment of head and neck tumours can severely impair the quality of life of the patients. In the current study we have investigated the early-to-late pathogenesis of the parotid gland after radiation. Also the ability to ameliorate the damage using pretreatment with adrenergic or muscarinic receptor agonists is studied. Rats were locally irradiated with or without i.p. pretreatment with phenylephrine (α-adrenoceptor agonist, 5 mg kg–1), isoproterenol (β-adrenoceptor agonist, 5 mg kg–1), pilocarpine (4 mg kg–1), methacholine (3.75 mg kg–1) (muscarinic receptor agonists) or methacholine plus phenylephrine. Parotid salivary flow rate, amylase secretion, the number of cells and gland histology were monitored sequentially up to 240 days postirradiation. The effects were described in 4 distinct phases. The first phase (0–10 days) was characterised by a rapid decline in flow rate without changes in amylase secretion or acinar cell number. The second phase (10–60 days) consists of a decrease in amylase secretion and is paralleled by acinar cell loss. Flow rate, amylase secretion and acinar cell numbers do not change in the third phase (60–120 days). The fourth phase (120–240 days) is determined by a further deterioration of gland function but an increase in acinar cell number, albeit with poor tissue morphology. All drug pretreatments used could reduce radiation effects in phase I and II. The protective effects were lost during phase IV, with the exception of methacholine plus phenylephrine pretreatment. The latter combination of drugs ameliorated radiation-damage throughout the entire follow-up time. The data show that combined pre-irradiation stimulation of muscarinic acetylcholine receptors with methacholine plus α-adrenoceptors with phenylephrine can reduce both early and late damage, possibly involving the PLC/PIP2 second messenger pathways. This opens perspectives for the development of clinical applicable methods for long-term sparing of parotid glands subjected to radiotherapy of head and neck cancer patients.

Keratinocyte Growth Factor Prevents Radiation Damage to Salivary Glands by Expansion of the Stem/Progenitor Pool

Irradiation of salivary glands during radiotherapy treatment of patients with head and neck cancer evokes persistent hyposalivation. This results from depletion of stem cells, which renders the gland incapable of replenishing saliva to produce acinar cells. The aim of this study was to investigate whether it is possible to expand the salivary gland stem/progenitor cell population, thereby preventing acinar cell depletion and subsequent gland dysfunction after irradiation. To induce cell proliferation, keratinocyte growth factor (ΔN23‐KGF, palifermin) was administered to C57BL/6 mice for 4 days before and/or after local irradiation of salivary glands. Salivary gland vitality was quantified by in vivo saliva flow rates, morphological measurements, and a newly developed in vitro salisphere progenitor/stem cell assay. Irradiation of salivary glands led to a pronounced reduction in the stem cells of the tissues, resulting in severe hyposalivation and a reduced number of acinar cells. ΔN23‐KGF treatment for 4 days before irradiation indeed induced salivary gland stem/progenitor cell proliferation, increasing the stem and progenitor cell pool. This did not change the relative radiation sensitivity of the stem/progenitor cells, but, as a consequence, an absolute higher number of stem/progenitor cells and acinar cells survived after radiation. Postirradiation treatment with ΔN23‐KGF also improved gland function, and this effect was much more pronounced in ΔN23‐KGF pretreated animals. Post‐treatment with ΔN23‐KGF seemed to act through accelerated expansion of the pool of progenitor/stem cells that survived the irradiation treatment. Overall, our data indicate that ΔN23‐KGF is a promising drug to enhance the number of salivary gland progenitor/stem cells and consequently prevent radiation‐induced hyposalivation.

Unexpected changes of rat cervical spinal cord tolerance caused by inhomogeneous dose distributions.

PURPOSE The effects of dose distribution on dose-effect relationships have been evaluated and, from this, iso-effective doses (ED(50)) established. METHODS AND MATERIALS Wistar rats were irradiated on the cervical spinal cord with single doses of unmodulated protons (150 MeV) to obtain sharp lateral penumbras, using the shoot-through technique, which employs the plateau of the depth-dose profile rather than the Bragg peak. Two types of inhomogeneous dose distributions have been administered: (1) 2 4-mm fields with 8- or 12-mm spacing between the center of the fields (referred to as split-field) were irradiated with variable single doses and (2) cervical spinal cord was irradiated with various combinations of relatively low doses to a large volume (20 mm) combined with high doses to a small volume (4 mm) (referred to as bath and shower). The endpoint for estimating the dose-response relationships was paralysis of the fore or hind limbs. RESULTS The split-field experiments (2 x 4 mm) showed a shift in the dose-response curves, giving significant higher ED(50) values of 45.4 Gy and 41.6 Gy for 8- and 12-mm spacing, respectively, compared with the ED(50) of 24.9 Gy for the single 8 mm (same total tissue volume irradiated). These values were closer to the ED(50) for a single 4-mm field of 53.7 Gy. The bath and shower experiments showed a large decrease of the ED(50) values from 15-22 Gy when compared with the 4-mm single field, even with a bath dose as low as 4 Gy. There were no histologic changes found in the low dose bath regions of the spinal cord at postmortem. CONCLUSIONS Not only the integral irradiated volume is a determining factor for the ED(50) of rat cervical spinal cord, but also the shape of the dose distribution is of great importance. The high ED(50) values of a small region or shower (4 mm) decreases significantly when the adjacent tissue is irradiated with a subthreshold dose (bath), even as low as 4 Gy. The significant shift to lower ED(50) values for induction of paralysis of the limbs by adding a low-dose bath was not accompanied by changes in histologic lesions. These observations may have implications for the interpretation of complex treatment plans and normal tissue complication probability in intensity-modulated radiotherapy.