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Volume effects and region-dependent radiosensitivity of the parotid gland
Int. J. Radiation Oncology Biol. Phys., Vol. 62, No. 4, pp. 1090 –1095, 2005 Copyright © 2005 Elsevier Inc. Printed in the USA. All rights reserved 0360-3016/05/$–see front matter
doi:10.1016/j.ijrobp.2004.12.035
CLINICAL INVESTIGATION
Head and Neck
VOLUME EFFECTS AND REGION-DEPENDENT RADIOSENSITIVITY OF THE PAROTID GLAND ANTONIUS W. T. KONINGS, PH.D.,* FEMMY COTTELEER,* HETTE FABER,* PETER HARM MEERTENS, PH.D.,† AND ROB P. COPPES, PH.D.*†
VAN
LUIJK, PH.D.,†
*Department of Radiation and Stress Cell Biology, University of Groningen, Groningen, The Netherlands; † Department of Radiation Oncology, University Hospital Groningen, Groningen, The Netherlands Purpose: To detect volume effects and possible regional differences in radiosensitivity of the rat parotid gland. Methods and Materials: Parotid glands of male albino Wistar rats were locally X-irradiated, with collimators with conformal radiation portals used to supply 100% volume and 50% cranial/caudal partial volumes. High-resolution magnetic resonance imaging was used to provide the outlines of the parotid glands. Single doses of up to 40 Gy were applied, and the effects on saliva secretion, measured with the aid of miniaturized Lashley cups, were followed up to 365 days after the irradiation. Results: Under conditions of equal mean absorbed doses and small variations in dose distribution, a pertinent volume effect was observed for late but not for early radiation damage. The late effects were different for the cranial part as compared with the caudal part of the parotid gland. The reduction in flow rate was much more severe after irradiation in the cranial part. After a single dose of 30 Gy, the reductions in flow rates were approximately 65% and 25% for the cranial and caudal parts, respectively. At that dose, no saliva flow was observed after irradiation of 100% of the gland. Conclusion: From the rat model studies presented, it is concluded that late radiation damage after partial irradiation of parotid glands shows region-dependent volume effects. This finding is expected to be relevant to the radiosensitivity of human salivary glands, and it implies that the predictive power of the mean dose concept in radiotherapeutic practice is limited. The finding of region-dependent late radiation damage also challenges the basic assumptions of most current normal tissue complication probability models for parotid gland function. © 2005 Elsevier Inc. Normal tissue damage, Parotid gland salivary secretion, Volume effects, Regional radiosensitivity, Secondary damage.
Irradiation for head and neck malignancies often results in reduced salivary output and altered salivary composition, leading to irreversible and distressing oral complaints by the treated patients (1). By 1 week after fractionated radiotherapy, salivary output is already severely reduced (1, 2). This acute radiation effect is enigmatic because the saliva-producing cells have a slow turnover, as is known from studies with rodents (3). The maximum dose delivered to the tumor during a course of radiotherapy is generally limited by the tolerance dose of healthy normal tissues. This limitation of normal tissue tolerance is usually addressed by posing constraints on the dose–volume histogram (DVH). One of the means of limiting radiation damage to healthy
tissues is to reduce the irradiated volume (4). The effect of volume reduction mainly depends on the anatomic structure of the organ. It might be anticipated that, when independent units of function in an organ (e.g., acini in salivary glands) have a parallel organization, irradiation of a small part of the organ has less effect in terms of function loss than when the organ has a serial anatomic organization. In the latter case, damage to one substructure disables the entire organ (e.g., in the spinal cord). Volume studies on lung (5, 6), spinal cord (7), and parotid gland (this report) of the rat are ongoing in our laboratories. In the literature (8), volume effects and differences in sensitivity between regions have already been suggested for mouse lung. With new radiation methods, including conformal and intensity-modulated radiation therapy (IMRT) techniques, a
Reprint requests to: Antonius W. T. Konings, Ph.D., Department of Radiation and Stress Cell Biology, University of Groningen, Building 3215, 5th Floor, Ant. Deusinglaan 1, 9717 BM Groningen, The Netherlands. Tel: (⫹31) 50-3180716; Fax: (⫹31) 50-3138784; E-mail: [email protected] This study was financed by the Proton Therapy Project of the
University of Groningen (Centrale Beleids Ruimte). Acknowledgments—The authors thank Judith Roesink, Chris Terhaard, Niels Raaijmakers, and Arjan Vissink for their fruitful comments on the manuscript. Received Dec 3, 2004, and in revised form Dec 22, 2004. Accepted for publication Dec 23, 2004.
INTRODUCTION
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Region-dependent radiosensitivity of the parotid gland
high dose is administered to only a small part of the salivary glands, positioned close to the tumor, while the rest of the healthy tissue receives a low dose or no dose at all. Therefore, it is important to have profound knowledge of dose distributions and dose–volume relationships in healthy tissue and the possible region-dependent radiosensitivity of the tissue. The aim of the current study was to investigate dose– volume effects on rat parotid gland function (flow rate) and to examine possible regional differences in radiosensitivity. To be sure that an altered reduction in the flow of saliva after radiation treatment can be fully attributed to the primary radiation insult to the gland itself and not to indirect effects of radiation damage to nongland tissue (e.g., oral, pharyngeal, or esophageal mucosa) lying within the radiation field (9), careful planning of the radiation portals is important. In the case of partial irradiation of salivary glands, the accuracy of the volume determination and the precise distribution of dose are even more important. We designed collimators with conformal radiation portals based on outlines of the gland obtained by high-resolution magnetic imaging (MRI) (10). Equal doses could be delivered to the 50% cranial and 50% caudal volumes. By using this method, region-dependent radiosensitivity in the parotid gland could be shown. METHODS AND MATERIALS Animals Male albino Wistar rats (Hsd/Cpb: WU) weighing 230 –250 g and 9 –10 weeks old (Harlan-CPB, Rijswijk, The Netherlands) were housed in polycarbonate cages (6 rats per cage) randomly placed in the racks, under a 14 hour/10 hour light/dark cycle, 2 weeks before the start of the experiments. Food (RHM-B, Hope Farms, Woerden, The Netherlands) and water was given ad libitum. All experiments were carried out in agreement with the Netherlands Experiments on Animals Act (1977) and the European Convention for the Protection of Vertebrates used for Experimental Purpose (Strasbourg, 18.III.1986) and met the standards required by the United Kingdom Coordinating Committee on Cancer Research 1998 guidelines.
Parotid gland irradiation Both glands were irradiated. The rats were placed in a radiation holder hanging on a positioning rod by their upper incisors (for details see Cotteller et al. (10)). Before positioning, the rats were anesthetized with an i.p. injection of Rompun (xylazine; Bayer AG, Leverkusen, Germany) plus Ketalar (S-ketamine; Pfizer BV, Capelle aan de IJssel, The Netherlands), allowing at least 30 min for anesthesia. For collimation, 3-mm lead collimators were used, resulting in conformal radiation beams for 100%, 50% cranial, or 50% caudal irradiation, as described earlier. Irradiation of the cranial 50% of the gland involved the ventral lobe and part of the dorsal lobe, whereas the caudal 50% included the lateral lobe and part of the dorsal lobe. A protocol for absolute dosimetry was designed, and relative dose measurements were performed. From the three-dimensional (3D) topography data and the 3D dose distributions, DVHs were determined. The designed setup showed that irradiation of the small parotid volumes could be performed with high accuracy (10).
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The irradiation holder was placed on a platform (pin-holes construction) attached to the beam exit window (bayonet-ring construction) of the X-ray tube. As a result, the position of the rat in the radiation beam was fixed. The platform was designed so that when the irradiation holder with the rat is placed on it, the central beam axis coincides with the z axis. In this way, the divergence of the beam is minimized. Irradiation was performed by an orthovoltage X-ray machine (Mueller MG 300; Philips, Eindhoven, The Netherlands) operated at 200-kV high voltage and 15-mA tube current. The beam was filtered by 0.5 mm Cu ⫹ 0.5 mm Al. The Al-filter was placed at the beam-exit side of the tube housing. This results in a first half value layer of 1.0 mm Cu. To obtain an acceptable field homogeneity and percentage dose distribution and to obtain a dose rate that allowed a single irradiation up to 50 Gy within 25 min, a focal spot to skin distance of 213 mm was chosen. During dose measurements and animal irradiation, the tube output was monitored with a 0.6-cm3 monitor ionization chamber (PTW “Farmer” chamber, B30001; PTW, Freiburg, Germany) connected to an electrometer (PTW Unidos-E 10008), which was placed inside the beam, just outside the portal projections. Detailed information on the 3D topography and the 3D dose distribution resulting in DVHs can be found elsewhere (10). Doses of 10, 15, 20, and 30 Gy were applied for the 100% and 50% volumes and additionally 40 Gy for the 50% volumes. For each experimental group a total of 6 – 8 rats were used.
Collection of saliva Saliva samples of both left and right parotid gland were collected simultaneously under isoflurane/O2 anesthesia by means of miniaturized Lasley cups (11). The cups were placed on the orifices of both parotid glands. Saliva was collected for 30 min after stimulation with 2 mg/kg pilocarpine administered s.c. (at t ⫽ 0 and t ⫽ 15 min). Saliva was collected in preweighed, ice-cooled plastic tubes 4 days before and 10, 30, 60, 120, 180, 240, 300, and 360 days after irradiation. As a parameter of gland function, saliva flow rates were determined. The total volume of saliva secreted was estimated by weight, assuming the specific gravity of saliva to be 1.0 g/cm3. The saliva flow rate (L/min⫺3) was calculated from the collecting time and volume, and expressed as the percentage of the value before irradiation (⫾ SEM).
RESULTS Figure 1 shows the flow rate in time after 100% volume irradiation with different doses of X-rays. The flow rate before treatment (100%) was 11.1 ⫾ 0.5 L/min. A rapid decline in function was already seen within 10 days after irradiation with a moderate single dose of 10 Gy. The degree of this acute damage is dose dependent, rising to 55% function loss after a single dose of 30 Gy. At Day 240, almost no saliva was excreted anymore after that dose. This decline in flow is comparable to earlier observations from our laboratory (12). When under the same conditions only 50% of the gland was irradiated, the result in terms of function loss was clearly dependent on the region of irradiation, as can be seen in Fig. 2. Irradiation of the 50% cranial volume with a single dose of 30 Gy resulted in much more late damage in gland function than irradiation of the caudal 50%.
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Fig. 1. Changes in parotid flow rate up to 365 days after local X-irradiation of the whole (100%) gland with different doses. Data are expressed as the percentage ⫾ SEM of the pretreatment value.
A dose– effect relationship for early effects (first 30 days after irradiation) is shown in Fig. 3. The effect for the cranial and the caudal radiations is comparable, and no clear region-dependent radiosensitivity is present. For late effects a completely different picture emerges, as can be seen in Fig. 4. A difference in radiosensitivity was observed between the cranial and caudal parts of the parotid gland for all doses examined. Irradiation of the 50% cranial volume resulted in more late damage in gland function than irradiation of the caudal 50% at the different X-ray exposures. DISCUSSION In radiobiologic textbooks (13), one can find descriptions of the definition of volume effect. However, defining “volume
Fig. 2. Region-dependent radiosensitivity of the parotid gland after partial X-irradiation with 30 Gy. Changes in flow rate were measured up to 365 days after the irradiation for the caudal and cranial 50% volumes. The 100% volume data are taken from Fig. 1 for reasons of comparison. The data are expressed as the percentage ⫾ SEM of the pretreatment value.
Volume 62, Number 4, 2005
Fig. 3. Dose– effect relationship for acute radiation damage on parotid gland function after total and partial X-irradiation. Gland function is expressed as the mean flow rate (⫾ SEM) during the first 30 days (area under the curve analysis) after X-irradiation with different doses delivered to the 50% cranial or 50% caudal volume and compared with the effect on the 100% volume. The mean flow rate of nonirradiated rats was approximately 110% of the flow rate at Day 0.
effect” only as the dependence of damage on volume is of limited practical use: almost all partial irradiations will show this effect. We propose a more restricted definition of the volume effect. Here, we define volume effect after partial irradiation of an organ or tissue as damage that is not proportional to the irradiated volume. For the healthy parotid gland, it is assumed that an equal and homogeneous distribution of saliva production takes place over the entire volume. When the radiation damage is more than proportional, the volume effect is described as positive, whereas a negative
Fig. 4. Dose– effect relationships for late radiation damage on parotid gland function after total and partial X-irradiation. Gland function is expressed as the mean flow rate (⫾ SEM) during the last 2 months (Months 11 and 12) after X-irradiation with different doses delivered to the 50% cranial or 50% caudal volume and compared with the effect on the 100% volume (area under the curve analysis). The mean flow rate of nonirradiated rats was approximately 130% of the flow rate at Day 0.
Region-dependent radiosensitivity of the parotid gland
volume effect corresponds with less-than-proportional damage. It is suggested that secondary tissue reactions in the noor low-dose exposed parts might be the cause of the volume effects. As can be seen in Fig. 5A, the parotid gland showed no volume effect for acute radiation damage when 50% of the volume was irradiated. For late effects, a positive volume effect was observed after 50% cranial irradiation. When the caudal 50% was irradiated, a tendency toward a negative volume effect was observed. Early radiation damage to the gland is probably caused by disturbed receptor-associated signaling pathways in the membrane of acinar cells (14), and loss of aquaporin-5 water channels (15). Because the radiation dose delivered to each part of the gland is the same (10), and the distribution of acini in the gland is homogeneous (unpublished observations), as expected, both halves respond more or less similarly. The volume effect is zero or close to zero. This indicates that at this stage, secondary damage in no- or low-dose exposed tissue does not play a major role. The slow turnover of acinar cells (3) might be a major explanation of this phenomenon. The expression of late radiation damage, however, is clearly dependent on volume. After cranial irradiation, secondary radiation effects seem to seriously affect stimulated parotid saliva flow. The interpretation of this late effect might be as follows. During the postirradiation period, damage to blood vessels, ducts, and neurons in the irradiated part will develop. The proper functioning of the shielded parts of the gland depends on these entities. Therefore, primary radiation damage in the irradiated parts might lead to secondary damage in the unirradiated parts of the gland and subsequently to an increased lack of function (positive volume effect). Irradiating the caudal part leads to
Fig. 5. Volume effects for acute (A) and late (B) radiation injury on parotid gland function. For the 50% volumes, it is indicated whether more or less than proportional damage has taken place (positive or negative volume effect) or that the damage is proportional to the 100% volume (no volume effect). Data are derived from Figs. 3 and 4.
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less damage than might be expected from direct irradiation effects only, which might be explained by compensation of function in the nonirradiated cranial part. It is assumed that the function of the cranial part is more or less independent from the caudal part (lateral lobe), and its saliva production might increase after irradiation, leading to the negative volume effect. Ongoing pathohistologic research in our laboratory will shed more light on the proposed mechanism of action. A situation of damage compensation, as suggested here, is often expected after radiotherapy for head and neck cancer but is difficult to prove because the volume of irradiated salivary tissue is either not exactly defined or unknown (16, 17). In many clinical treatments in the head and neck region, the dose administered to the parotid glands is distributed inhomogeneously over the organ. In treatment planning for such irradiations, the inhomogeneity is reflected by the shape of the DVH of the parotid glands. The differences in median effective dose for the salivary gland as obtained by Eisbruch et al. (18) and Roesink et al. (19) might be due to different DVH shapes but also to spatial differences in the treatment plans. To correlate the damage to the parotid gland and the dose distribution, one needs a model that characterizes the dose distribution (e.g., the mean dose). At present, the clinical knowledge of salivary gland dose–response is based on studies reporting the mean dose–response for a group of patients (18 –20). One should, however, keep in mind that in current DVHs and in the mean dose model, the spatial information is lost. For a comparison of rival plans, whereby the aim is to spare two or at least one of the parotid glands, the plan with the lowest mean dose to the parotids is usually considered the best plan as far as the parotids are concerned. The results of the current volume studies show, however, that spatial information like the differences in sensitivity between the cranial and the caudal part (because of secondary damage) of the parotid gland in rats is relevant. One might expect this finding also to be important for humans, in whom for instance a difference between the medial and lateral lobe might be present because of secondary damage. These considerations imply that the predictive power of the mean dose model is limited. For clinical practice, it is often convenient to define the flow rate at a certain time point after irradiation beneath a certain level (threshold, remaining function, cut-off point) as nontolerable and convert the flow rate data to a binary response (“yes” or “no” complication). Although rather arbitrary, a clinical complication might then be defined as a stimulated parotid flow rate, at a certain moment in time, beneath a certain threshold (percent function of preradiotherapy flow rate). The Radiation Therapy Oncology Group/ European Organization for Research and Treatment of Cancer (21) suggests a threshold of 25%. In Figs. 6 and 7, it is shown that the choice of a threshold value is very decisive for the shape of normal tissue complication probability (NTCP) curves.
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Volume 62, Number 4, 2005
Fig. 6. Normal tissue complication probability (NTCP) for 40% remaining function after total and partial X-irradiation of the parotid gland. Each data point represents the fraction of the number of animals that showed less than 40% remaining function at a certain dose of X-rays.
Fig. 7. Normal tissue complication probability (NTCP) for 75% remaining function after total and partial X-irradiation of the parotid gland. Each data point represents the fraction of the number of animals that showed less than 75% remaining function at a certain dose of X-rays.
When we accept 60% function loss (40% threshold), no early normal tissue complications are seen (Fig. 6A) after both types of 50% volume irradiations, not even at 40 Gy. When, however, the threshold is increased and the acceptance is lowered to only 25% function loss (75% threshold, Fig. 7A), early radiation complications are obvious. For late effects (approximately 1 year after irradiation), no complications for caudal irradiations are registered up to 40 Gy at the 40% threshold, whereas a 75% threshold leads to clear complications (Fig. 6B and 7B). These examples illustrate the importance of the introduction of a threshold of acceptance for the construction and use of an NTCP curve. In all cases, the regional difference in parotid radiosensitivity after cranial and caudal irradiation is present. Currently applied NTCP models base their assumptions on the cell survival concept. Early radiation damage is, however, probably not caused by cell death (14) but rather by membrane damage to the acinar cells; the cells do not disappear. Cell killing for late damage is an important factor. The overall effect is, however, obscured (positive or negative volume effects) by secondary damage and by com-
pensation reactions in no- and low-dose irradiated fields, depending on the regional distribution of the dose in the organ. CONCLUSION Partial irradiation of the parotid gland of the rat leads to varying late radiation damage, depending on the region that has been exposed. It is suggested that the regional difference in radiosensitivity is caused by secondary effects. The function of a nonirradiated region might be hampered (positive volume effect) when that region is structurally dependent (e.g., ducts, blood flow) on the irradiated part but might be improved (negative volume effect, caused by compensation mechanisms) when it is independent from the irradiated part. There is no reason to believe that the observed regional difference in gland radiosensitivity will not occur after partial irradiation of the glands during radiotherapy of patients with head and neck cancer. This notion implies that the predictive power, in radiotherapeutic practice, of the mean dose concept is limited and challenges the basic assumptions of most current NTCP models for parotid gland function.
REFERENCES 1. Vissink A, Jansma J, Spijkervet FKL, et al. Oral sequelae of head and neck radiotherapy. Crit Rev Oral Biol Med 2003;14:199 – 212. 2. Burlage FR, Coppes RP, Meertens H, et al. Parotid and submandibular/sublingual flow during high dose radiotherapy. Radiother Oncol 2001;61:271–274. 3. Schwartz-Arad D, Arber L, Arber N, et al. The rat parotid gland. A renewing cell population. J Anat 1988;161:143– 151. 4. Hopewell JW, Trott K-R. Volume effects in radiobiology as applied to radiotherapy. Radiother Oncol 2000;56:283–288. 5. Wiegman EM, Meertens H, Konings AWT, et al. Loco-regional
differences in pulmonary function and density after partial rat lung irradiation. Radiother Oncol 2003;69:11–19. 6. Jiresova A, Cotteleer F, van Goor H, et al. Dose-region effects in partial irradiation of the lung. Radiother Oncol 2002; 64(Suppl. 1):192. 7. Bijl HP, van Luijk P, Coppes RP, et al. Changes of rat cervical spinal cord tolerance caused by inhomogeneous dose distributions. Int J Radiat Oncol Biol Phys 2003;57: 274 –281. 8. Tucker SL, Liao ZX, Travis EL. Estimation of the spatial distribution of target cell for radiation pneumonitis in the mouse lung. Int J Radiat Oncol Biol Phys 1997;38:1359 –1370.
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9. Konings AWT, Vissink A, Coppes RP. Comments on: Extended-term effects of head and neck irradiation in a rodent. Eur J Cancer 2002;38:851– 852. 10. Cotteleer F, Faber H, Konings AWT, et al. Three-dimensional dose distribution for partial irradiation of rat parotid glands with 200kV X-rays. Int J Radiat Biol 2003;79:689 –700. 11. Vissink A, ’s-Gravenmade EJ, Konings AWT, et al. The adaptation of the Lashley cup for use in rat saliva collection. Arch Oral Biol 1989;34:577–578. 12. Coppes RP, Zeilstra LJW, Kampinga HH, et al. Early to late sparing of radiation damage to the parotid gland by adrenergic and muscarinic receptor agonists. Br J Cancer 2001;2038: 1055–1063. 13. Steel GG. Basic clinical radiobiology. 2nd ed. London: Arnold; 1997. 14. Konings AWT, Coppes RP, Vissink A. On the mechanism of salivary gland radiosensitivity. Int J Radiat Oncol Biol Phys 2005;1187–1194. 15. Takagi K, Yamaguchi K, Sakurai T, et al. Secretion of saliva in X-irradiated rat submandibular glands. Radiat Res 2003; 159:351–360.
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16. Valdez IH, Atkinson JC, Ship JA, et al. Major salivary gland function in patients with radiation-induced xerostomia: Flow rates and sialochemistry. Int J Radiat Oncol Biol Phys 1993; 25:41– 47. 17. Liu RP, Fleming TJ, Toth BB, et al. Salivary flow rates in patients with head and neck cancer 0.5 to 25 years after radiotherapy. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 1990;70:724 –729. 18. Eisbruch A, Ten Haken RK, Hyungjin MK, et al. Dose, volume, and function relationships in parotid salivary glands following conformal and intensity-modulated irradiation of head and neck cancer. Int J Radiat Oncol Biol Phys 1999;45: 577–587. 19. Roesink JM, Moerland MA, Batterman JJ, et al. Quantitative dose-volume response analysis of changes in parotid gland function after radiotherapy in the head and neck region. Int J Radiat Oncol Biol Phys 2001;51:938 –946. 20. Eisbruch A, Ship JA, Hyungjin MK, et al. Partial irradiation of the parotid gland. Sem Radiat Oncol 2001;11:234 –239. 21. LENT SOMA tables. Radiother Oncol 1995;35:17– 60.
doi:10.1016/j.ijrobp.2004.12.035
CLINICAL INVESTIGATION
Head and Neck
VOLUME EFFECTS AND REGION-DEPENDENT RADIOSENSITIVITY OF THE PAROTID GLAND ANTONIUS W. T. KONINGS, PH.D.,* FEMMY COTTELEER,* HETTE FABER,* PETER HARM MEERTENS, PH.D.,† AND ROB P. COPPES, PH.D.*†
VAN
LUIJK, PH.D.,†
*Department of Radiation and Stress Cell Biology, University of Groningen, Groningen, The Netherlands; † Department of Radiation Oncology, University Hospital Groningen, Groningen, The Netherlands Purpose: To detect volume effects and possible regional differences in radiosensitivity of the rat parotid gland. Methods and Materials: Parotid glands of male albino Wistar rats were locally X-irradiated, with collimators with conformal radiation portals used to supply 100% volume and 50% cranial/caudal partial volumes. High-resolution magnetic resonance imaging was used to provide the outlines of the parotid glands. Single doses of up to 40 Gy were applied, and the effects on saliva secretion, measured with the aid of miniaturized Lashley cups, were followed up to 365 days after the irradiation. Results: Under conditions of equal mean absorbed doses and small variations in dose distribution, a pertinent volume effect was observed for late but not for early radiation damage. The late effects were different for the cranial part as compared with the caudal part of the parotid gland. The reduction in flow rate was much more severe after irradiation in the cranial part. After a single dose of 30 Gy, the reductions in flow rates were approximately 65% and 25% for the cranial and caudal parts, respectively. At that dose, no saliva flow was observed after irradiation of 100% of the gland. Conclusion: From the rat model studies presented, it is concluded that late radiation damage after partial irradiation of parotid glands shows region-dependent volume effects. This finding is expected to be relevant to the radiosensitivity of human salivary glands, and it implies that the predictive power of the mean dose concept in radiotherapeutic practice is limited. The finding of region-dependent late radiation damage also challenges the basic assumptions of most current normal tissue complication probability models for parotid gland function. © 2005 Elsevier Inc. Normal tissue damage, Parotid gland salivary secretion, Volume effects, Regional radiosensitivity, Secondary damage.
Irradiation for head and neck malignancies often results in reduced salivary output and altered salivary composition, leading to irreversible and distressing oral complaints by the treated patients (1). By 1 week after fractionated radiotherapy, salivary output is already severely reduced (1, 2). This acute radiation effect is enigmatic because the saliva-producing cells have a slow turnover, as is known from studies with rodents (3). The maximum dose delivered to the tumor during a course of radiotherapy is generally limited by the tolerance dose of healthy normal tissues. This limitation of normal tissue tolerance is usually addressed by posing constraints on the dose–volume histogram (DVH). One of the means of limiting radiation damage to healthy
tissues is to reduce the irradiated volume (4). The effect of volume reduction mainly depends on the anatomic structure of the organ. It might be anticipated that, when independent units of function in an organ (e.g., acini in salivary glands) have a parallel organization, irradiation of a small part of the organ has less effect in terms of function loss than when the organ has a serial anatomic organization. In the latter case, damage to one substructure disables the entire organ (e.g., in the spinal cord). Volume studies on lung (5, 6), spinal cord (7), and parotid gland (this report) of the rat are ongoing in our laboratories. In the literature (8), volume effects and differences in sensitivity between regions have already been suggested for mouse lung. With new radiation methods, including conformal and intensity-modulated radiation therapy (IMRT) techniques, a
Reprint requests to: Antonius W. T. Konings, Ph.D., Department of Radiation and Stress Cell Biology, University of Groningen, Building 3215, 5th Floor, Ant. Deusinglaan 1, 9717 BM Groningen, The Netherlands. Tel: (⫹31) 50-3180716; Fax: (⫹31) 50-3138784; E-mail: [email protected] This study was financed by the Proton Therapy Project of the
University of Groningen (Centrale Beleids Ruimte). Acknowledgments—The authors thank Judith Roesink, Chris Terhaard, Niels Raaijmakers, and Arjan Vissink for their fruitful comments on the manuscript. Received Dec 3, 2004, and in revised form Dec 22, 2004. Accepted for publication Dec 23, 2004.
INTRODUCTION
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Region-dependent radiosensitivity of the parotid gland
high dose is administered to only a small part of the salivary glands, positioned close to the tumor, while the rest of the healthy tissue receives a low dose or no dose at all. Therefore, it is important to have profound knowledge of dose distributions and dose–volume relationships in healthy tissue and the possible region-dependent radiosensitivity of the tissue. The aim of the current study was to investigate dose– volume effects on rat parotid gland function (flow rate) and to examine possible regional differences in radiosensitivity. To be sure that an altered reduction in the flow of saliva after radiation treatment can be fully attributed to the primary radiation insult to the gland itself and not to indirect effects of radiation damage to nongland tissue (e.g., oral, pharyngeal, or esophageal mucosa) lying within the radiation field (9), careful planning of the radiation portals is important. In the case of partial irradiation of salivary glands, the accuracy of the volume determination and the precise distribution of dose are even more important. We designed collimators with conformal radiation portals based on outlines of the gland obtained by high-resolution magnetic imaging (MRI) (10). Equal doses could be delivered to the 50% cranial and 50% caudal volumes. By using this method, region-dependent radiosensitivity in the parotid gland could be shown. METHODS AND MATERIALS Animals Male albino Wistar rats (Hsd/Cpb: WU) weighing 230 –250 g and 9 –10 weeks old (Harlan-CPB, Rijswijk, The Netherlands) were housed in polycarbonate cages (6 rats per cage) randomly placed in the racks, under a 14 hour/10 hour light/dark cycle, 2 weeks before the start of the experiments. Food (RHM-B, Hope Farms, Woerden, The Netherlands) and water was given ad libitum. All experiments were carried out in agreement with the Netherlands Experiments on Animals Act (1977) and the European Convention for the Protection of Vertebrates used for Experimental Purpose (Strasbourg, 18.III.1986) and met the standards required by the United Kingdom Coordinating Committee on Cancer Research 1998 guidelines.
Parotid gland irradiation Both glands were irradiated. The rats were placed in a radiation holder hanging on a positioning rod by their upper incisors (for details see Cotteller et al. (10)). Before positioning, the rats were anesthetized with an i.p. injection of Rompun (xylazine; Bayer AG, Leverkusen, Germany) plus Ketalar (S-ketamine; Pfizer BV, Capelle aan de IJssel, The Netherlands), allowing at least 30 min for anesthesia. For collimation, 3-mm lead collimators were used, resulting in conformal radiation beams for 100%, 50% cranial, or 50% caudal irradiation, as described earlier. Irradiation of the cranial 50% of the gland involved the ventral lobe and part of the dorsal lobe, whereas the caudal 50% included the lateral lobe and part of the dorsal lobe. A protocol for absolute dosimetry was designed, and relative dose measurements were performed. From the three-dimensional (3D) topography data and the 3D dose distributions, DVHs were determined. The designed setup showed that irradiation of the small parotid volumes could be performed with high accuracy (10).
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The irradiation holder was placed on a platform (pin-holes construction) attached to the beam exit window (bayonet-ring construction) of the X-ray tube. As a result, the position of the rat in the radiation beam was fixed. The platform was designed so that when the irradiation holder with the rat is placed on it, the central beam axis coincides with the z axis. In this way, the divergence of the beam is minimized. Irradiation was performed by an orthovoltage X-ray machine (Mueller MG 300; Philips, Eindhoven, The Netherlands) operated at 200-kV high voltage and 15-mA tube current. The beam was filtered by 0.5 mm Cu ⫹ 0.5 mm Al. The Al-filter was placed at the beam-exit side of the tube housing. This results in a first half value layer of 1.0 mm Cu. To obtain an acceptable field homogeneity and percentage dose distribution and to obtain a dose rate that allowed a single irradiation up to 50 Gy within 25 min, a focal spot to skin distance of 213 mm was chosen. During dose measurements and animal irradiation, the tube output was monitored with a 0.6-cm3 monitor ionization chamber (PTW “Farmer” chamber, B30001; PTW, Freiburg, Germany) connected to an electrometer (PTW Unidos-E 10008), which was placed inside the beam, just outside the portal projections. Detailed information on the 3D topography and the 3D dose distribution resulting in DVHs can be found elsewhere (10). Doses of 10, 15, 20, and 30 Gy were applied for the 100% and 50% volumes and additionally 40 Gy for the 50% volumes. For each experimental group a total of 6 – 8 rats were used.
Collection of saliva Saliva samples of both left and right parotid gland were collected simultaneously under isoflurane/O2 anesthesia by means of miniaturized Lasley cups (11). The cups were placed on the orifices of both parotid glands. Saliva was collected for 30 min after stimulation with 2 mg/kg pilocarpine administered s.c. (at t ⫽ 0 and t ⫽ 15 min). Saliva was collected in preweighed, ice-cooled plastic tubes 4 days before and 10, 30, 60, 120, 180, 240, 300, and 360 days after irradiation. As a parameter of gland function, saliva flow rates were determined. The total volume of saliva secreted was estimated by weight, assuming the specific gravity of saliva to be 1.0 g/cm3. The saliva flow rate (L/min⫺3) was calculated from the collecting time and volume, and expressed as the percentage of the value before irradiation (⫾ SEM).
RESULTS Figure 1 shows the flow rate in time after 100% volume irradiation with different doses of X-rays. The flow rate before treatment (100%) was 11.1 ⫾ 0.5 L/min. A rapid decline in function was already seen within 10 days after irradiation with a moderate single dose of 10 Gy. The degree of this acute damage is dose dependent, rising to 55% function loss after a single dose of 30 Gy. At Day 240, almost no saliva was excreted anymore after that dose. This decline in flow is comparable to earlier observations from our laboratory (12). When under the same conditions only 50% of the gland was irradiated, the result in terms of function loss was clearly dependent on the region of irradiation, as can be seen in Fig. 2. Irradiation of the 50% cranial volume with a single dose of 30 Gy resulted in much more late damage in gland function than irradiation of the caudal 50%.
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Fig. 1. Changes in parotid flow rate up to 365 days after local X-irradiation of the whole (100%) gland with different doses. Data are expressed as the percentage ⫾ SEM of the pretreatment value.
A dose– effect relationship for early effects (first 30 days after irradiation) is shown in Fig. 3. The effect for the cranial and the caudal radiations is comparable, and no clear region-dependent radiosensitivity is present. For late effects a completely different picture emerges, as can be seen in Fig. 4. A difference in radiosensitivity was observed between the cranial and caudal parts of the parotid gland for all doses examined. Irradiation of the 50% cranial volume resulted in more late damage in gland function than irradiation of the caudal 50% at the different X-ray exposures. DISCUSSION In radiobiologic textbooks (13), one can find descriptions of the definition of volume effect. However, defining “volume
Fig. 2. Region-dependent radiosensitivity of the parotid gland after partial X-irradiation with 30 Gy. Changes in flow rate were measured up to 365 days after the irradiation for the caudal and cranial 50% volumes. The 100% volume data are taken from Fig. 1 for reasons of comparison. The data are expressed as the percentage ⫾ SEM of the pretreatment value.
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Fig. 3. Dose– effect relationship for acute radiation damage on parotid gland function after total and partial X-irradiation. Gland function is expressed as the mean flow rate (⫾ SEM) during the first 30 days (area under the curve analysis) after X-irradiation with different doses delivered to the 50% cranial or 50% caudal volume and compared with the effect on the 100% volume. The mean flow rate of nonirradiated rats was approximately 110% of the flow rate at Day 0.
effect” only as the dependence of damage on volume is of limited practical use: almost all partial irradiations will show this effect. We propose a more restricted definition of the volume effect. Here, we define volume effect after partial irradiation of an organ or tissue as damage that is not proportional to the irradiated volume. For the healthy parotid gland, it is assumed that an equal and homogeneous distribution of saliva production takes place over the entire volume. When the radiation damage is more than proportional, the volume effect is described as positive, whereas a negative
Fig. 4. Dose– effect relationships for late radiation damage on parotid gland function after total and partial X-irradiation. Gland function is expressed as the mean flow rate (⫾ SEM) during the last 2 months (Months 11 and 12) after X-irradiation with different doses delivered to the 50% cranial or 50% caudal volume and compared with the effect on the 100% volume (area under the curve analysis). The mean flow rate of nonirradiated rats was approximately 130% of the flow rate at Day 0.
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volume effect corresponds with less-than-proportional damage. It is suggested that secondary tissue reactions in the noor low-dose exposed parts might be the cause of the volume effects. As can be seen in Fig. 5A, the parotid gland showed no volume effect for acute radiation damage when 50% of the volume was irradiated. For late effects, a positive volume effect was observed after 50% cranial irradiation. When the caudal 50% was irradiated, a tendency toward a negative volume effect was observed. Early radiation damage to the gland is probably caused by disturbed receptor-associated signaling pathways in the membrane of acinar cells (14), and loss of aquaporin-5 water channels (15). Because the radiation dose delivered to each part of the gland is the same (10), and the distribution of acini in the gland is homogeneous (unpublished observations), as expected, both halves respond more or less similarly. The volume effect is zero or close to zero. This indicates that at this stage, secondary damage in no- or low-dose exposed tissue does not play a major role. The slow turnover of acinar cells (3) might be a major explanation of this phenomenon. The expression of late radiation damage, however, is clearly dependent on volume. After cranial irradiation, secondary radiation effects seem to seriously affect stimulated parotid saliva flow. The interpretation of this late effect might be as follows. During the postirradiation period, damage to blood vessels, ducts, and neurons in the irradiated part will develop. The proper functioning of the shielded parts of the gland depends on these entities. Therefore, primary radiation damage in the irradiated parts might lead to secondary damage in the unirradiated parts of the gland and subsequently to an increased lack of function (positive volume effect). Irradiating the caudal part leads to
Fig. 5. Volume effects for acute (A) and late (B) radiation injury on parotid gland function. For the 50% volumes, it is indicated whether more or less than proportional damage has taken place (positive or negative volume effect) or that the damage is proportional to the 100% volume (no volume effect). Data are derived from Figs. 3 and 4.
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less damage than might be expected from direct irradiation effects only, which might be explained by compensation of function in the nonirradiated cranial part. It is assumed that the function of the cranial part is more or less independent from the caudal part (lateral lobe), and its saliva production might increase after irradiation, leading to the negative volume effect. Ongoing pathohistologic research in our laboratory will shed more light on the proposed mechanism of action. A situation of damage compensation, as suggested here, is often expected after radiotherapy for head and neck cancer but is difficult to prove because the volume of irradiated salivary tissue is either not exactly defined or unknown (16, 17). In many clinical treatments in the head and neck region, the dose administered to the parotid glands is distributed inhomogeneously over the organ. In treatment planning for such irradiations, the inhomogeneity is reflected by the shape of the DVH of the parotid glands. The differences in median effective dose for the salivary gland as obtained by Eisbruch et al. (18) and Roesink et al. (19) might be due to different DVH shapes but also to spatial differences in the treatment plans. To correlate the damage to the parotid gland and the dose distribution, one needs a model that characterizes the dose distribution (e.g., the mean dose). At present, the clinical knowledge of salivary gland dose–response is based on studies reporting the mean dose–response for a group of patients (18 –20). One should, however, keep in mind that in current DVHs and in the mean dose model, the spatial information is lost. For a comparison of rival plans, whereby the aim is to spare two or at least one of the parotid glands, the plan with the lowest mean dose to the parotids is usually considered the best plan as far as the parotids are concerned. The results of the current volume studies show, however, that spatial information like the differences in sensitivity between the cranial and the caudal part (because of secondary damage) of the parotid gland in rats is relevant. One might expect this finding also to be important for humans, in whom for instance a difference between the medial and lateral lobe might be present because of secondary damage. These considerations imply that the predictive power of the mean dose model is limited. For clinical practice, it is often convenient to define the flow rate at a certain time point after irradiation beneath a certain level (threshold, remaining function, cut-off point) as nontolerable and convert the flow rate data to a binary response (“yes” or “no” complication). Although rather arbitrary, a clinical complication might then be defined as a stimulated parotid flow rate, at a certain moment in time, beneath a certain threshold (percent function of preradiotherapy flow rate). The Radiation Therapy Oncology Group/ European Organization for Research and Treatment of Cancer (21) suggests a threshold of 25%. In Figs. 6 and 7, it is shown that the choice of a threshold value is very decisive for the shape of normal tissue complication probability (NTCP) curves.
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Fig. 6. Normal tissue complication probability (NTCP) for 40% remaining function after total and partial X-irradiation of the parotid gland. Each data point represents the fraction of the number of animals that showed less than 40% remaining function at a certain dose of X-rays.
Fig. 7. Normal tissue complication probability (NTCP) for 75% remaining function after total and partial X-irradiation of the parotid gland. Each data point represents the fraction of the number of animals that showed less than 75% remaining function at a certain dose of X-rays.
When we accept 60% function loss (40% threshold), no early normal tissue complications are seen (Fig. 6A) after both types of 50% volume irradiations, not even at 40 Gy. When, however, the threshold is increased and the acceptance is lowered to only 25% function loss (75% threshold, Fig. 7A), early radiation complications are obvious. For late effects (approximately 1 year after irradiation), no complications for caudal irradiations are registered up to 40 Gy at the 40% threshold, whereas a 75% threshold leads to clear complications (Fig. 6B and 7B). These examples illustrate the importance of the introduction of a threshold of acceptance for the construction and use of an NTCP curve. In all cases, the regional difference in parotid radiosensitivity after cranial and caudal irradiation is present. Currently applied NTCP models base their assumptions on the cell survival concept. Early radiation damage is, however, probably not caused by cell death (14) but rather by membrane damage to the acinar cells; the cells do not disappear. Cell killing for late damage is an important factor. The overall effect is, however, obscured (positive or negative volume effects) by secondary damage and by com-
pensation reactions in no- and low-dose irradiated fields, depending on the regional distribution of the dose in the organ. CONCLUSION Partial irradiation of the parotid gland of the rat leads to varying late radiation damage, depending on the region that has been exposed. It is suggested that the regional difference in radiosensitivity is caused by secondary effects. The function of a nonirradiated region might be hampered (positive volume effect) when that region is structurally dependent (e.g., ducts, blood flow) on the irradiated part but might be improved (negative volume effect, caused by compensation mechanisms) when it is independent from the irradiated part. There is no reason to believe that the observed regional difference in gland radiosensitivity will not occur after partial irradiation of the glands during radiotherapy of patients with head and neck cancer. This notion implies that the predictive power, in radiotherapeutic practice, of the mean dose concept is limited and challenges the basic assumptions of most current NTCP models for parotid gland function.
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differences in pulmonary function and density after partial rat lung irradiation. Radiother Oncol 2003;69:11–19. 6. Jiresova A, Cotteleer F, van Goor H, et al. Dose-region effects in partial irradiation of the lung. Radiother Oncol 2002; 64(Suppl. 1):192. 7. Bijl HP, van Luijk P, Coppes RP, et al. Changes of rat cervical spinal cord tolerance caused by inhomogeneous dose distributions. Int J Radiat Oncol Biol Phys 2003;57: 274 –281. 8. Tucker SL, Liao ZX, Travis EL. Estimation of the spatial distribution of target cell for radiation pneumonitis in the mouse lung. Int J Radiat Oncol Biol Phys 1997;38:1359 –1370.
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16. Valdez IH, Atkinson JC, Ship JA, et al. Major salivary gland function in patients with radiation-induced xerostomia: Flow rates and sialochemistry. Int J Radiat Oncol Biol Phys 1993; 25:41– 47. 17. Liu RP, Fleming TJ, Toth BB, et al. Salivary flow rates in patients with head and neck cancer 0.5 to 25 years after radiotherapy. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 1990;70:724 –729. 18. Eisbruch A, Ten Haken RK, Hyungjin MK, et al. Dose, volume, and function relationships in parotid salivary glands following conformal and intensity-modulated irradiation of head and neck cancer. Int J Radiat Oncol Biol Phys 1999;45: 577–587. 19. Roesink JM, Moerland MA, Batterman JJ, et al. Quantitative dose-volume response analysis of changes in parotid gland function after radiotherapy in the head and neck region. Int J Radiat Oncol Biol Phys 2001;51:938 –946. 20. Eisbruch A, Ship JA, Hyungjin MK, et al. Partial irradiation of the parotid gland. Sem Radiat Oncol 2001;11:234 –239. 21. LENT SOMA tables. Radiother Oncol 1995;35:17– 60.