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20 Microarray analysis to identify mechanisms of radiation-induced microvascular damage in normal tissues
$8
Monday, October 25, 2004
have shown that lower urethral doses were associated with less urinary symptoms when using an intraoperative treatment planning approach which more effectively constrains urethral doses. There is also ample evidence in the literature demonstrating that late rectal morbidity correlates strongly with the percentage of the rectal surface that received high doses such as >100 Gy. Meticulous attention to needle and seed placement in the operating room as well as intra-operative dose-volume histograms of normal tissue should reduce rectal doses and risks of toxicity to minimal levels. There is less evidence and conflicting reports in the literature as to the role of the dose to the neurovascular bundles and penile bulb and its impact on erectile function after prostate brachytherapy. While it is recommended to minimize dose to these structures it is difficult to conclusively prove that dose consistently has a direct effect on erectile function in this patient population. This may likely be due to the fact that erectile function is a multifactorial phenomenon depending upon may variables including the age of the patients, baseline function and the current medical co-morbidities of the patient.
Genetic approaches to assess normal tissue RT-sensitivity 18 Single nucleotide polymorphisms in TGF-~I and other candidate genes J. Alsner, C.N. Andreassen, J. Overgaard Aarhus University Hospital, Dept. Experimental Clinical Oncology, Aarhus, Denmark An overview is presented of the published data available, together with an update of our own analysis, on single nucleotide polymorphisms (SNPs) in candidate genes and their effect on the variability in normal tissue radiosensitivity. A number of recent studies have indicated that variations in genes related to DNA damage detection and repair, pro-fibrotic cytokines, modulators of extracellular matrix components and scavengers of reactive oxygen species affect phenomena like cancer susceptibility, in vitro radiosensitivity and risk of different fibrotic disorders. Although we are still far from having a complete understanding of the genetic variation that may underlie differences in normal tissue reactions when unselected cancer patients undergo radiotherapy, variations in genes related to the biological response to radiation injury seems to affect the individual risk of radiotherapy complications. Examples of this are the observed links between variations in the genes encoding ATM and TGF-L31 and the risk of subcutaneous fibrosis in breast cancer patients. Present data supports the assumption that the genetic component of clinical normal tissue radiosensitivity should be regarded as a complex trait dependent on the cumulative effect of variation in several genes. Furthermore, different models must be established for different clinical/biological endpoints. To fully address these questions, further studies in different areas such as whole genome screening, haplotype associations, and functional analysis of candidate SNPs are needed. Finally, carefully designed clinical studies with an accrual of very large numbers of patients (like the ESTRO GENEPI project) are of the outmost importance.
Symposia
19 Genes induced by IR E. Travis U. T. M.D. Anderson Cancer Center, Dept. of Experimental Rad. Onc., Box 66, Houston, TX, U.S.A. New cancer treatments are improving cure rates in some malignancies with an increase in long-term survivors but with the potential for a concomitant increase in treatment related complications. The pathogenesis of radiation induced tissue damage is complex with multiple pathways interacting with little known about inciting and perpetuating injuries, identity of key effector cells and mediators, mechanisms of remodelling of tissue architecture and the genes regulating the process. Current therapies aimed at limiting these response have had limited success in the clinic. Radiation induced tissue damage is, in part, genetically regulated but none of the genes involved are known which is at least partially because it is a complex polygenic trait making the use of only classical genetics to study these disorders tedious and inefficient and prohibitively expensive, requiring 3 to 5 years in the mouse just to identify chromosomal loci associated with a disease. A powerful approach that facilitates and accelerates gene discovery is to combine classical genetics with microarray technology which has numerous advantages over global gene expression studies e.g., identification of pathways involved in the response. This approach has been highly successful in identifying genes in other complex traits such as asthma and diabetes. Recently, using this approach, we identified candidate genes for radiation induced lung fibrosis including a family of transcription factors, KLF, the cell cycle checkpoint gene p21, and a novel gene, TFF2, none of which are obvious candidate genes. The use of microarray technology allows an efficient, objective, quantitative evaluation of genes in the QTL and the opportunity to identify potentially important genes that most likely would have escaped detection. 20 Microarray analysis to identify mechanisms of radiationinduced microvascular damage in normal tissues
J.J.C.M. Kruse 1,3,J.A.M. te Peele 1, N.S. Russell2, F.A. Stewart 1 1The Netherlands Cancer Institute, Department of Experimental Therapy, Amsterdam, The Netherlands 2The Netherlands Cancer Institute, Department of Radiotherapy, Amsterdam, The Netherlands 3Leiden University Medical Centre (LUMC), Division 5, Department of Toxicogenetica, Leiden, The Netherlands The purpose of the study is to identify changes in gene expression in irradiated tissue and to understand the transcriptional networks that underlie the development of radiation-induced microvascular damage in normal tissues. In order to provide that crucial understanding, cDNA microarray analysis was performed using amplified RNA isolated from irradiated and sham-irradiated mouse kidney and rectum at different time intervals (1-30 weeks) after irradiation. Semiquantitative PCR analysis was performed on a selection of genes to provide independent confirmation of microarray data. In the analysis of microarray data, the use of the error-model and combined information from self-self, straight colour and colour reverse experiments provided a quantitative basis for identifying statistical significant changes in gene expression. This approach substantially increased the reliability of the microarray results compared with the selection of an arbitrary fixed-threshold approach as a measure of differential expression.
Symposia
Monday, October 25, 2004 $9
A small percentage of the genes present on the arrays met the stringent criteria for differential expression that was applied in this analysis. The selected genes included regulators of vascular development and angiogenic remodelling (Jagged-I, KLF-5), as well as genes involved in inflammatory pathways (TSA-1). The present study demonstrated that the microarray approach is an effective tool in identifying candidate genes that were not previously known to be associated with molecular and cellular biological processes in normal tissues after irradiation. Further studies will be required to evaluate the functional role of the differentially expressed genes in the development of vascularmediated normal tissue injury.
Novel technology in radiotherapy 21 Tomotherapy: preliminary methodology for RT
experience
with
a
new
R. Ca/andrino. G.M. Cattaneo, S. Broggi, S. Molinel/i, C. Fiorino Istituto Scientifico San Raffaele, Servizio Fisica Sanitaria, Milano, Italy Commercial clinical helical Tomotherapy unit (Hi-Art 2 System) is a new modality for radiation treatments: it's the first treatment unit dedicated to intensity modulated radiotherapy (IMRT) with a fully integrated image-guided radiotherapy system with the on-board mega-voltage CT (MVCT) capability. The Tomotherapy system uses a 6 MV accelerator, a 64-leaf binary multileaf collimator and xenon image detector array mounted on a rotating slip-ring: the radiation is delivered in an helical way, obtained by concurrent gantry rotation and couch/patient travel; together, these components allow continuous intensity modulated rotational delivery of radiation with fan beam entry from 360-degree. Optimisation process in Tomotherapy represents a special challenge: pre-optimisation step, where the main free parameters (field size, pitch, modulation factor) are determined and beamlets pre-calculated; optimisation step where the dose constraints are selected and the plan optimised accordingly by using an iterative last-square minimisation technique and a post-optimisation step, where the dose distribution is finally recalculated to account dosimetric and mechanical MLC's characteristics (time leaf latency; Tongue-groove effect). Convolution/superpositJon (C/S) dose calculation is employed for ultimate dose calculation. The availability of a CT scanner on board allows a number of verification processes: a MVCT scan before the treatment can be fused with a planning CT scan to determine the correct setup of the patient. The CT detector signal during treatment could be used to reconstruct the effectively delivered dose to the patient to be compared with the expected one. The capability to reduce the uncertainty in the patient set-up and the ability of this IMRT technique to produce very large dose gradients and rapid dose falioff outside the target makes it desirable to reduce margins limiting the high dose volume in adjacent structures by offering the possibility to increase the biologically effective dose to the target. In this presentation different aspects of the Tomotherapy process will be summarised: commissioning test, QA procedures, treatment plans examples and clinical aspects will be described.
22 Robotic approaches in patient positioning: 4 weddings ... and a funeral? R. Ferrand Centre de Protonth6rapie d'Orsay, Orsay Cedex, France The major evolution in radiotherapy towards targeted irradiation and high conformation has led to a quest for precision at every step of the treatment process. And patient positioning is one of the crucial and hardest issues to solve since it depends on the patient himself: immobilisation, Iocalisation, displacement and error compensation .... Furthermore, although a precise patient positioning must also be fast (to minimise patient motion), speed and precision are usually incompatible, unless a reliable and automated procedure assists in reaching such an optimisation. For years, radiotherapy and robotics ignored each other, one in the medical world, the other mostly focused to industry (space, cars,...). As conformal therapy emerged, patient positioning became a "six degrees of freedom problem" as well as a "4D problem" (time dimension), for which robotics can bring elegant, fast, precise but also reliable answers, based on a strong industrial experience. Several examples, based on the use of industrial robots, have already proven the high potential of today's system adapted to a medical use. From the specification required today in patient positioning, we will consider the different fields (target Iocalisation, motion calculation and correction, real time tracking, automation) where the links between the robotics concepts and radiotherapy will certainly be reinforced in the future, with as final goal a safe, highly precise, fully automated, patient positioning. 23 Cyberknife: user's point of view P. Francescon Ospedale San Bortolo, Medical Physics, Vicenza, Italy CyberKnife is an innovative radiosurgery device based on a compact linear accelerator mounted on a robotic arm, and on a x-ray imaging system allowing non-isocentric, frameless operations. The non-isocentric approach is the main characteristic which allows highly conformal isodose shapes; it is possible thanks to a robotic arm with 6 degrees of freedom. The Linac source is positioned at 80 cm from the virtual isocenter; 100 positions can be assumed by the source on a sphere centered on this point, and from each position 12 directions can be reached, leading to 1200 different beams in total. Not all these directions will probably be used, but it is thanks to such a flexibility, and to the different weighting of the beams, that highly conformal shapes can be achieved. The Linac is a compact, 6MV unit with circular collimators ranging from 5 to 60mm. Compared to conventional stereotactic radiosurgery systems, the CyberKnife provides enhanced ability to avoid critical structures, thanks to highly conformal dose distribution, dose fractionation (allowed by reliable relocation) and potential to target multiple tumors at different locations during a single treatment. The localization system is based on the comparison between a set of digitally reconstructed radiographs (DRRs), generated from a CT scan, and actual x-ray pictures taken from orthogonal directions. From the comparison of the two pairs of images it is possible to calculate six parameters that describe the translation and rotation errors. After an initial alignment which is performed by moving the treatment couch, the system tracks further movements and accounts for them by redefining the frame of reference - that is, patient movements are corrected by correcting the position of the robot.
Monday, October 25, 2004
have shown that lower urethral doses were associated with less urinary symptoms when using an intraoperative treatment planning approach which more effectively constrains urethral doses. There is also ample evidence in the literature demonstrating that late rectal morbidity correlates strongly with the percentage of the rectal surface that received high doses such as >100 Gy. Meticulous attention to needle and seed placement in the operating room as well as intra-operative dose-volume histograms of normal tissue should reduce rectal doses and risks of toxicity to minimal levels. There is less evidence and conflicting reports in the literature as to the role of the dose to the neurovascular bundles and penile bulb and its impact on erectile function after prostate brachytherapy. While it is recommended to minimize dose to these structures it is difficult to conclusively prove that dose consistently has a direct effect on erectile function in this patient population. This may likely be due to the fact that erectile function is a multifactorial phenomenon depending upon may variables including the age of the patients, baseline function and the current medical co-morbidities of the patient.
Genetic approaches to assess normal tissue RT-sensitivity 18 Single nucleotide polymorphisms in TGF-~I and other candidate genes J. Alsner, C.N. Andreassen, J. Overgaard Aarhus University Hospital, Dept. Experimental Clinical Oncology, Aarhus, Denmark An overview is presented of the published data available, together with an update of our own analysis, on single nucleotide polymorphisms (SNPs) in candidate genes and their effect on the variability in normal tissue radiosensitivity. A number of recent studies have indicated that variations in genes related to DNA damage detection and repair, pro-fibrotic cytokines, modulators of extracellular matrix components and scavengers of reactive oxygen species affect phenomena like cancer susceptibility, in vitro radiosensitivity and risk of different fibrotic disorders. Although we are still far from having a complete understanding of the genetic variation that may underlie differences in normal tissue reactions when unselected cancer patients undergo radiotherapy, variations in genes related to the biological response to radiation injury seems to affect the individual risk of radiotherapy complications. Examples of this are the observed links between variations in the genes encoding ATM and TGF-L31 and the risk of subcutaneous fibrosis in breast cancer patients. Present data supports the assumption that the genetic component of clinical normal tissue radiosensitivity should be regarded as a complex trait dependent on the cumulative effect of variation in several genes. Furthermore, different models must be established for different clinical/biological endpoints. To fully address these questions, further studies in different areas such as whole genome screening, haplotype associations, and functional analysis of candidate SNPs are needed. Finally, carefully designed clinical studies with an accrual of very large numbers of patients (like the ESTRO GENEPI project) are of the outmost importance.
Symposia
19 Genes induced by IR E. Travis U. T. M.D. Anderson Cancer Center, Dept. of Experimental Rad. Onc., Box 66, Houston, TX, U.S.A. New cancer treatments are improving cure rates in some malignancies with an increase in long-term survivors but with the potential for a concomitant increase in treatment related complications. The pathogenesis of radiation induced tissue damage is complex with multiple pathways interacting with little known about inciting and perpetuating injuries, identity of key effector cells and mediators, mechanisms of remodelling of tissue architecture and the genes regulating the process. Current therapies aimed at limiting these response have had limited success in the clinic. Radiation induced tissue damage is, in part, genetically regulated but none of the genes involved are known which is at least partially because it is a complex polygenic trait making the use of only classical genetics to study these disorders tedious and inefficient and prohibitively expensive, requiring 3 to 5 years in the mouse just to identify chromosomal loci associated with a disease. A powerful approach that facilitates and accelerates gene discovery is to combine classical genetics with microarray technology which has numerous advantages over global gene expression studies e.g., identification of pathways involved in the response. This approach has been highly successful in identifying genes in other complex traits such as asthma and diabetes. Recently, using this approach, we identified candidate genes for radiation induced lung fibrosis including a family of transcription factors, KLF, the cell cycle checkpoint gene p21, and a novel gene, TFF2, none of which are obvious candidate genes. The use of microarray technology allows an efficient, objective, quantitative evaluation of genes in the QTL and the opportunity to identify potentially important genes that most likely would have escaped detection. 20 Microarray analysis to identify mechanisms of radiationinduced microvascular damage in normal tissues
J.J.C.M. Kruse 1,3,J.A.M. te Peele 1, N.S. Russell2, F.A. Stewart 1 1The Netherlands Cancer Institute, Department of Experimental Therapy, Amsterdam, The Netherlands 2The Netherlands Cancer Institute, Department of Radiotherapy, Amsterdam, The Netherlands 3Leiden University Medical Centre (LUMC), Division 5, Department of Toxicogenetica, Leiden, The Netherlands The purpose of the study is to identify changes in gene expression in irradiated tissue and to understand the transcriptional networks that underlie the development of radiation-induced microvascular damage in normal tissues. In order to provide that crucial understanding, cDNA microarray analysis was performed using amplified RNA isolated from irradiated and sham-irradiated mouse kidney and rectum at different time intervals (1-30 weeks) after irradiation. Semiquantitative PCR analysis was performed on a selection of genes to provide independent confirmation of microarray data. In the analysis of microarray data, the use of the error-model and combined information from self-self, straight colour and colour reverse experiments provided a quantitative basis for identifying statistical significant changes in gene expression. This approach substantially increased the reliability of the microarray results compared with the selection of an arbitrary fixed-threshold approach as a measure of differential expression.
Symposia
Monday, October 25, 2004 $9
A small percentage of the genes present on the arrays met the stringent criteria for differential expression that was applied in this analysis. The selected genes included regulators of vascular development and angiogenic remodelling (Jagged-I, KLF-5), as well as genes involved in inflammatory pathways (TSA-1). The present study demonstrated that the microarray approach is an effective tool in identifying candidate genes that were not previously known to be associated with molecular and cellular biological processes in normal tissues after irradiation. Further studies will be required to evaluate the functional role of the differentially expressed genes in the development of vascularmediated normal tissue injury.
Novel technology in radiotherapy 21 Tomotherapy: preliminary methodology for RT
experience
with
a
new
R. Ca/andrino. G.M. Cattaneo, S. Broggi, S. Molinel/i, C. Fiorino Istituto Scientifico San Raffaele, Servizio Fisica Sanitaria, Milano, Italy Commercial clinical helical Tomotherapy unit (Hi-Art 2 System) is a new modality for radiation treatments: it's the first treatment unit dedicated to intensity modulated radiotherapy (IMRT) with a fully integrated image-guided radiotherapy system with the on-board mega-voltage CT (MVCT) capability. The Tomotherapy system uses a 6 MV accelerator, a 64-leaf binary multileaf collimator and xenon image detector array mounted on a rotating slip-ring: the radiation is delivered in an helical way, obtained by concurrent gantry rotation and couch/patient travel; together, these components allow continuous intensity modulated rotational delivery of radiation with fan beam entry from 360-degree. Optimisation process in Tomotherapy represents a special challenge: pre-optimisation step, where the main free parameters (field size, pitch, modulation factor) are determined and beamlets pre-calculated; optimisation step where the dose constraints are selected and the plan optimised accordingly by using an iterative last-square minimisation technique and a post-optimisation step, where the dose distribution is finally recalculated to account dosimetric and mechanical MLC's characteristics (time leaf latency; Tongue-groove effect). Convolution/superpositJon (C/S) dose calculation is employed for ultimate dose calculation. The availability of a CT scanner on board allows a number of verification processes: a MVCT scan before the treatment can be fused with a planning CT scan to determine the correct setup of the patient. The CT detector signal during treatment could be used to reconstruct the effectively delivered dose to the patient to be compared with the expected one. The capability to reduce the uncertainty in the patient set-up and the ability of this IMRT technique to produce very large dose gradients and rapid dose falioff outside the target makes it desirable to reduce margins limiting the high dose volume in adjacent structures by offering the possibility to increase the biologically effective dose to the target. In this presentation different aspects of the Tomotherapy process will be summarised: commissioning test, QA procedures, treatment plans examples and clinical aspects will be described.
22 Robotic approaches in patient positioning: 4 weddings ... and a funeral? R. Ferrand Centre de Protonth6rapie d'Orsay, Orsay Cedex, France The major evolution in radiotherapy towards targeted irradiation and high conformation has led to a quest for precision at every step of the treatment process. And patient positioning is one of the crucial and hardest issues to solve since it depends on the patient himself: immobilisation, Iocalisation, displacement and error compensation .... Furthermore, although a precise patient positioning must also be fast (to minimise patient motion), speed and precision are usually incompatible, unless a reliable and automated procedure assists in reaching such an optimisation. For years, radiotherapy and robotics ignored each other, one in the medical world, the other mostly focused to industry (space, cars,...). As conformal therapy emerged, patient positioning became a "six degrees of freedom problem" as well as a "4D problem" (time dimension), for which robotics can bring elegant, fast, precise but also reliable answers, based on a strong industrial experience. Several examples, based on the use of industrial robots, have already proven the high potential of today's system adapted to a medical use. From the specification required today in patient positioning, we will consider the different fields (target Iocalisation, motion calculation and correction, real time tracking, automation) where the links between the robotics concepts and radiotherapy will certainly be reinforced in the future, with as final goal a safe, highly precise, fully automated, patient positioning. 23 Cyberknife: user's point of view P. Francescon Ospedale San Bortolo, Medical Physics, Vicenza, Italy CyberKnife is an innovative radiosurgery device based on a compact linear accelerator mounted on a robotic arm, and on a x-ray imaging system allowing non-isocentric, frameless operations. The non-isocentric approach is the main characteristic which allows highly conformal isodose shapes; it is possible thanks to a robotic arm with 6 degrees of freedom. The Linac source is positioned at 80 cm from the virtual isocenter; 100 positions can be assumed by the source on a sphere centered on this point, and from each position 12 directions can be reached, leading to 1200 different beams in total. Not all these directions will probably be used, but it is thanks to such a flexibility, and to the different weighting of the beams, that highly conformal shapes can be achieved. The Linac is a compact, 6MV unit with circular collimators ranging from 5 to 60mm. Compared to conventional stereotactic radiosurgery systems, the CyberKnife provides enhanced ability to avoid critical structures, thanks to highly conformal dose distribution, dose fractionation (allowed by reliable relocation) and potential to target multiple tumors at different locations during a single treatment. The localization system is based on the comparison between a set of digitally reconstructed radiographs (DRRs), generated from a CT scan, and actual x-ray pictures taken from orthogonal directions. From the comparison of the two pairs of images it is possible to calculate six parameters that describe the translation and rotation errors. After an initial alignment which is performed by moving the treatment couch, the system tracks further movements and accounts for them by redefining the frame of reference - that is, patient movements are corrected by correcting the position of the robot.