- Email: [email protected]
33 invited DNA microarrays and normal tissue morbidity
$8 Sunday, 10 June 2001
32
invited
Modification of acute radio(chemo)therapy effects in squamous e p i t h e l i a b y k e r a t i n o c y t e g r o w t h factor w. D6rr Klinik und Poliklinik for Strahlentherapie und Radioonkologie, Medizinische Fakult~t, 01307 Dresden, Germany Keratinocyte Growth Factor (KGF) is a member of the fibroblast growth factor family. KGF is produced by mesenchymal cells, predominantly fibroblasts. Target cells are epithelial cells in a variety of tissues. Hence, KGF is a mediator of the mesenchymal-epithelial communication and a regulator of tissue homeostasis in epithelia. Systemic administration of KGF in animal models induces stimulation of proliferation and modulation of migration and differentiation processes in squamous epithelia. This results in a temporary increase in cell numbers and epithelial thickness. Radiation exposure of epithelia causes an imbalance between cell production and cell loss, which in consequence results in progressive cell depletion and eventually complete denudation. The most detailed normal tissue study with KGF was performed in the mouse tongue model, where mucosal ulceration was analysed as the clinically relevant endpoint. The protocols included combination with single radiation doses, fractionated irradiation (5 daily fractions of 3 Gy) or single dose radiochemotherapy (cisDDP, 5-FU). KGF was administered in a variety of protocols. A significant reduction in oral mucosal toxicity by KGF-administration was observed, with an increase in isoeffective doses by a factor of 1.7-2.3. The most pronounced effect was seen when KGF was given after single dose or during fractionated radiation exposure. Additional studies in standard cell culture, reconstituted oral epithelium and tissue sections demonstrated increased cell production and changes in epithelial differentiation processes. The latter may result in a reduction of cell loss. A hypothetical explanation of the experimental results,k which is currently validated in histological studies, is that the radiation-induced cell depletion in KGF-treated epithelium is counteracted by the additional cells until tissue restoration by surviving cells is efficient. Hence ulcerative responses occur at a significantly lower frequency after a given radiation dose. In conclusion, KGF may represent an effective approach for amelioration of radiation effects in oral, gastrointestinal and cutaneous epithelia. Results from a clinical pilot study indicate that KGF is well tolerated and effective in humans. In studies with tumour cell lines or xenograffs, no or only a very minor effects of KGF was observed. Hence, selective amelioration of radio(chemo)therapy-induced toxicity can be expected. 33
invited
DNA microarrays and normal tissue morbidity M. McKay Peter Macca//um Cancer Institute, Research Division, Melbourne, Australia DNA microarrays (also known as DNA 'Chips') are a subtype of recently developed innovative technologies with considerable potential to impact radiobiological research and the practice of oncology. DNA microarrays are capable of simultaneously measuring gene expression ('activity') of thousands of genes, providing a global profile of patterns of gene expression characteristic of different physiological or disease states. Already, DNA microarrays have been successfully employed to provide novel molecular expression 'signatures' of gene activity in tumours. The procedure thus provides an avenue for the identification and development of new forms of molecular classification, diagnostics and prognostication. DNA microarrays also have application in high throughput genotyping and mutation analysis. Different types of DNA microarrays analysis have been developed: these will be briefly reviewed and contextualised in relation to complementary technologies with the potential to synergise with array approaches. Occasionally, cancer patients manifest unexpected adverse reactions to radiotherapy (RT). There is considerable evidence for a genetic basis to such ionising radiation (IR) sensitivity. Hence, using DNA microarrays, it may be possible to detect a gene expression 'signature' characteristic of radiosensitive phenotypes, by analysing normal tissues from RT patients who have experienced normal tissue morbidity. We developed a tissue bank of biological samples from cancer patients who have had adverse RT reactions. These samples and their appropriate controls (patients with otherwise similar phenotypes who did not experience adverse normal tissue reactions) have been-examined using DNA microarrays and patterns of gene activity are being sought which track with RT sensitivity. Such experiments may also lead to the development of testable hypotheses regarding different gene pathways which are abnormal in an individual case, allowing, for example, identification of loss-of-function mutations resulting in reduced expression of candidate radiation response genes. This could thereby facilitate identification of the molecular defect(s) in individual radiosensitive cases. These studies have also uncovered many genes previously unknown to form part of the stress response to IR, and should they be fully
successful have the potential to contribute to the individualisation of cancer therapy. 34
Quantification of late complications after radiation therapy H. Juno 1, H.-P. Beck-Bomholdt 1, V. Svoboda 1, W. Alberti 2, T. Herrmann3 1institute of Biophysics and Radiobiology, University of Hamburg, Hamburg, Germany 2Department of Radiotherapy and Radiooncology, University of Hamburg, Hamburg, Germany 3Department of Radiotherapy and Radiooncology, Technical University, Dresden, Germany (1) The risk of developing late complications after radiation therapy is generally believed to level off with time after irradiation. (2) Sixty-three patients treated by radical surgery and irradiation for rectal carcinoma were subjected to an unconventional sandwich therapy. A considerable proportion of these patients developed severe late complications (Svoboda et al., Radiother. Oncol. 53: 177-187, 1999). (3) Data on late complications were obtained for eight different organs with a follow-up of up to ten years. For the various organs, the percentage of patients being free from late complications, plotted as a function of time after start of radiation therapy, was adequately described by exponential regression. From the fit, the parameter pa was obtained, which is the percentage of patients at risk in a given year developing a complication in a given organ during that year. The rate pa remained about constant with time. (4) The results indicated that the hazard of developing late complications after irradiation remained about constant for many years. The reason is that the number of patients at risk decreased in a similar pattern as that for late complications leading to a constant risk per year. - From numerous data sets collected from published reports, three types of kinetics for the occurrence of late effects after radiotherapy were identified. Type 1: purely exponential kinetics; Type 2: exponential kinetics, the slope of which decreased exponentially with time; Type 3: curves composed of two components; a fast initial decline followed by an exponential decrease. For each kind of kinetics, provided dose distribution is not too heterogeneous, the incidence of late effects appears to occur at exponential or approximately exponential kinetics, even many years after treatment. (5) Because of the probably lifelong risk of developing late complications, it might become necessary to change frequency and duration of follow-up after radiation therapy and to extend patient's information on longterm radiation risks. (Supported by Roggenbuck-Stiftung, Hamburg). 35 C e l l u l a r and molecular characterization o f
late radiation enteritis M. Vozenin-Brotons1, A. Francois 1, J. Saboufin2, P. Lasser3, C. HaieMedef4, A. Lusinchi4, S. Antoun 3, J. Aiguepersel 1,5, D. Math~5 T. Girinsky1, 4 I lPSN/IGR, Unitd METSI, Villejuif, France 21GR, Pathology, Villejuif, France 31GR, Surgery, Villejuif, France 41GR, Radiotherapy, Villejuif, France 51PSN, DPHD/SARAM, Villejuif, France Introduction : Normal tissues might receive significant doses of ionizing radiation during therapeutic or accidental irradiation. In particular, the highly radiosensitive gastrointestinal tract is affected during the treatment of pelvic cancers. Some patients developed late radiation enteritis and bowel obstruction mostly caused by fibrosis. Fibrosis is typically associated with mesenchymal cells hyperplasia, tissue disorganization and transmural fibrillar collagen deposition. Fibrosis is thought to develop as a result of pathological tissue repair processes, but cellular and molecular mechanisms that underlie late radiation enteritis are not well defined. M&M : For this study we selected 13 patients treated in the Institut Gustave Roussy that developed late radiation enteritis after treatment for rectal cancers (RT[50-55Gy] Surg/-CT) or gynecological cancers (Surg RT[45Gy] CurieT[15Gy] /-CT). Pathology slides from resected ileum were used for histopathologioal and immunohistochemioal studies. Surgical tissue from 2 of the 13 patients were used for mRNA studies. Hybrid selection on high density cDNA arrays (CIontech) was used to characterize the mRNA expression profile of 1200 genes from late radiation enteritis as compared to a healthy ileum. Results : Histological abnormalities were scored after HES and revealed epithelial surface damages, lamina propria inflammation, thickness of muscularis propria, sub-mucosal and serosal fibrosis and vascular hyalinisation. Sirius red and Masson's trichrome staining confirmed the transmural deposition of collagenous extracellular matrix probably synthetized by activated
32
invited
Modification of acute radio(chemo)therapy effects in squamous e p i t h e l i a b y k e r a t i n o c y t e g r o w t h factor w. D6rr Klinik und Poliklinik for Strahlentherapie und Radioonkologie, Medizinische Fakult~t, 01307 Dresden, Germany Keratinocyte Growth Factor (KGF) is a member of the fibroblast growth factor family. KGF is produced by mesenchymal cells, predominantly fibroblasts. Target cells are epithelial cells in a variety of tissues. Hence, KGF is a mediator of the mesenchymal-epithelial communication and a regulator of tissue homeostasis in epithelia. Systemic administration of KGF in animal models induces stimulation of proliferation and modulation of migration and differentiation processes in squamous epithelia. This results in a temporary increase in cell numbers and epithelial thickness. Radiation exposure of epithelia causes an imbalance between cell production and cell loss, which in consequence results in progressive cell depletion and eventually complete denudation. The most detailed normal tissue study with KGF was performed in the mouse tongue model, where mucosal ulceration was analysed as the clinically relevant endpoint. The protocols included combination with single radiation doses, fractionated irradiation (5 daily fractions of 3 Gy) or single dose radiochemotherapy (cisDDP, 5-FU). KGF was administered in a variety of protocols. A significant reduction in oral mucosal toxicity by KGF-administration was observed, with an increase in isoeffective doses by a factor of 1.7-2.3. The most pronounced effect was seen when KGF was given after single dose or during fractionated radiation exposure. Additional studies in standard cell culture, reconstituted oral epithelium and tissue sections demonstrated increased cell production and changes in epithelial differentiation processes. The latter may result in a reduction of cell loss. A hypothetical explanation of the experimental results,k which is currently validated in histological studies, is that the radiation-induced cell depletion in KGF-treated epithelium is counteracted by the additional cells until tissue restoration by surviving cells is efficient. Hence ulcerative responses occur at a significantly lower frequency after a given radiation dose. In conclusion, KGF may represent an effective approach for amelioration of radiation effects in oral, gastrointestinal and cutaneous epithelia. Results from a clinical pilot study indicate that KGF is well tolerated and effective in humans. In studies with tumour cell lines or xenograffs, no or only a very minor effects of KGF was observed. Hence, selective amelioration of radio(chemo)therapy-induced toxicity can be expected. 33
invited
DNA microarrays and normal tissue morbidity M. McKay Peter Macca//um Cancer Institute, Research Division, Melbourne, Australia DNA microarrays (also known as DNA 'Chips') are a subtype of recently developed innovative technologies with considerable potential to impact radiobiological research and the practice of oncology. DNA microarrays are capable of simultaneously measuring gene expression ('activity') of thousands of genes, providing a global profile of patterns of gene expression characteristic of different physiological or disease states. Already, DNA microarrays have been successfully employed to provide novel molecular expression 'signatures' of gene activity in tumours. The procedure thus provides an avenue for the identification and development of new forms of molecular classification, diagnostics and prognostication. DNA microarrays also have application in high throughput genotyping and mutation analysis. Different types of DNA microarrays analysis have been developed: these will be briefly reviewed and contextualised in relation to complementary technologies with the potential to synergise with array approaches. Occasionally, cancer patients manifest unexpected adverse reactions to radiotherapy (RT). There is considerable evidence for a genetic basis to such ionising radiation (IR) sensitivity. Hence, using DNA microarrays, it may be possible to detect a gene expression 'signature' characteristic of radiosensitive phenotypes, by analysing normal tissues from RT patients who have experienced normal tissue morbidity. We developed a tissue bank of biological samples from cancer patients who have had adverse RT reactions. These samples and their appropriate controls (patients with otherwise similar phenotypes who did not experience adverse normal tissue reactions) have been-examined using DNA microarrays and patterns of gene activity are being sought which track with RT sensitivity. Such experiments may also lead to the development of testable hypotheses regarding different gene pathways which are abnormal in an individual case, allowing, for example, identification of loss-of-function mutations resulting in reduced expression of candidate radiation response genes. This could thereby facilitate identification of the molecular defect(s) in individual radiosensitive cases. These studies have also uncovered many genes previously unknown to form part of the stress response to IR, and should they be fully
successful have the potential to contribute to the individualisation of cancer therapy. 34
Quantification of late complications after radiation therapy H. Juno 1, H.-P. Beck-Bomholdt 1, V. Svoboda 1, W. Alberti 2, T. Herrmann3 1institute of Biophysics and Radiobiology, University of Hamburg, Hamburg, Germany 2Department of Radiotherapy and Radiooncology, University of Hamburg, Hamburg, Germany 3Department of Radiotherapy and Radiooncology, Technical University, Dresden, Germany (1) The risk of developing late complications after radiation therapy is generally believed to level off with time after irradiation. (2) Sixty-three patients treated by radical surgery and irradiation for rectal carcinoma were subjected to an unconventional sandwich therapy. A considerable proportion of these patients developed severe late complications (Svoboda et al., Radiother. Oncol. 53: 177-187, 1999). (3) Data on late complications were obtained for eight different organs with a follow-up of up to ten years. For the various organs, the percentage of patients being free from late complications, plotted as a function of time after start of radiation therapy, was adequately described by exponential regression. From the fit, the parameter pa was obtained, which is the percentage of patients at risk in a given year developing a complication in a given organ during that year. The rate pa remained about constant with time. (4) The results indicated that the hazard of developing late complications after irradiation remained about constant for many years. The reason is that the number of patients at risk decreased in a similar pattern as that for late complications leading to a constant risk per year. - From numerous data sets collected from published reports, three types of kinetics for the occurrence of late effects after radiotherapy were identified. Type 1: purely exponential kinetics; Type 2: exponential kinetics, the slope of which decreased exponentially with time; Type 3: curves composed of two components; a fast initial decline followed by an exponential decrease. For each kind of kinetics, provided dose distribution is not too heterogeneous, the incidence of late effects appears to occur at exponential or approximately exponential kinetics, even many years after treatment. (5) Because of the probably lifelong risk of developing late complications, it might become necessary to change frequency and duration of follow-up after radiation therapy and to extend patient's information on longterm radiation risks. (Supported by Roggenbuck-Stiftung, Hamburg). 35 C e l l u l a r and molecular characterization o f
late radiation enteritis M. Vozenin-Brotons1, A. Francois 1, J. Saboufin2, P. Lasser3, C. HaieMedef4, A. Lusinchi4, S. Antoun 3, J. Aiguepersel 1,5, D. Math~5 T. Girinsky1, 4 I lPSN/IGR, Unitd METSI, Villejuif, France 21GR, Pathology, Villejuif, France 31GR, Surgery, Villejuif, France 41GR, Radiotherapy, Villejuif, France 51PSN, DPHD/SARAM, Villejuif, France Introduction : Normal tissues might receive significant doses of ionizing radiation during therapeutic or accidental irradiation. In particular, the highly radiosensitive gastrointestinal tract is affected during the treatment of pelvic cancers. Some patients developed late radiation enteritis and bowel obstruction mostly caused by fibrosis. Fibrosis is typically associated with mesenchymal cells hyperplasia, tissue disorganization and transmural fibrillar collagen deposition. Fibrosis is thought to develop as a result of pathological tissue repair processes, but cellular and molecular mechanisms that underlie late radiation enteritis are not well defined. M&M : For this study we selected 13 patients treated in the Institut Gustave Roussy that developed late radiation enteritis after treatment for rectal cancers (RT[50-55Gy] Surg/-CT) or gynecological cancers (Surg RT[45Gy] CurieT[15Gy] /-CT). Pathology slides from resected ileum were used for histopathologioal and immunohistochemioal studies. Surgical tissue from 2 of the 13 patients were used for mRNA studies. Hybrid selection on high density cDNA arrays (CIontech) was used to characterize the mRNA expression profile of 1200 genes from late radiation enteritis as compared to a healthy ileum. Results : Histological abnormalities were scored after HES and revealed epithelial surface damages, lamina propria inflammation, thickness of muscularis propria, sub-mucosal and serosal fibrosis and vascular hyalinisation. Sirius red and Masson's trichrome staining confirmed the transmural deposition of collagenous extracellular matrix probably synthetized by activated