48 DNA damage response in radioresistant gliomas

Invited Speakers

March 12 - 15

impact on the radiotherapy community, as it will drive (either increase or reduce) the complexity of technology in routine use. M a t e r i a l a n d M e t h o d s : Thus, it is of interest to determine the precision/accuracy of the models and measurements actually required to meet clinical goals, including the point of diminishing returns for interventional strategies. We would like to know the degree of complexity actually warranted for a given patient (in modeling, measurement and intervention), making the most efficient use of modern motion modeling tools and techniques. Studies are now underway in several institutions to investigate motion models of varying complexity and assess changes in treatment evaluation metrics associated with their use. Additional studies are aimed at assessment of the time points at which updates to patient and motion models become available to determine the relative levels of benefit of plan modification later in treatment. A major goal of such investigations will be the development of the most accurate models possible of patient motion, position, and related changes, and then to study how reduction of this information to simpler levels of complexity would impact our ability to maintain maximal gains in accuracy of optimized and delivered treatments. R e s u l t s : Within this context, examples of the use of 4D models for radiotherapy of lung tumors with respect to the determination of clinical endpoints will be discussed. For example, the requirements for acquiring and using such complex data could be much different for determination of (e.g.) the mean dose to normal lung over the course or treatment, as opposed to (e.g.) tracking the dose that some small sub-volume of a shrinking tumor might receive on a daily basis. Similarly, depending upon the clinical goal, the marginal benefit of performing additional measurements on each patient over the course of treatment could reach the point of diminishing returns with respect to a) changes in the "relevant" patient model, and b) the ability to still be able to make any changes in treatment with the limited dose yet deliverable that would make a difference in outcome. C o n c l u s i o n : In addition to better tailoring overall treatments, investigations of this sort wilt help determine how much complexity (in modeling, measurement and intervention) is actually beneficial for a given patient, thus helping to establish the most efficient use of advanced in-room imaging resources within the radiotherapy community. 46 MICRO-ARRAY OUTCOME

PROFILES

IN

PREDICTING

TUMOR

H. Bartelink The Netherlands Cancer Institute, Amsterdam, The Netherlands Not received 47 RADIOTHERAPY OF B R E A S T NORMAL TISSUE REACTIONS?

CANCER

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PREDICTING

J. Overgaard Department of Experimental Clinical Oncology, Aarhus University Hospital, Aarhus C, Denmark The risk of normal tissue is frequently the limiting factor when deciding the dose of radiotherapy. Especially the late and often progressive morbidity constitute a problem, and the risk of such morbidity must be balanced with the potential benefit of the cancer treatment. It is estimated that approx. 3-4% of all irradiated patients will suffer from severe morbidity and even morbidity. The increasing knowledge on late effects have consequently given attention to modern techniques of precision radiotherapy (e.g. IMRT) which due to better focused physical dose distribution may reduce the problem. Late radiation morbidity is organ and tissue related, but in general is it considered to be dependent of the volume and total dose and the number of fractions, in such a way that larger doses per fraction causes a relative increase in morbidity when compared with the probability of tumor control. So far have the attempt to modify the therapeutic ratio thus been to reduce the physical dose of radiation to the organs at risk, and optimize the fractionation schedule by hyperfractionation. When it comes to individual risk factors, may these be related to certain comorbidities, but otherwise has it been the assumption that

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almost all patients in principle have the same risk and sensitivity for developing late morbidity. Earlier attempt to estimate in vitro radiosensitivity have indicated some potential individual variation, but the methods used have been too crude for predictive clinical use, except for patients with rare genetic disorders (e.g. ataxia telangiectasia). The use of new biological genomic techniques together with an increased understanding of variations in genetic function and expressions have, however, opened a new dimension in our understanding of the pathogenesis of late effects. Results from cDNA gene expression have identified radiation induced expression profiles with distinct patterns related to sensitivity, but unfortunately this will require in vitro radiation of living cells. More importantly are there strong indications that polymorphisms in specific candidate genes may be related to both general radiosensitivity as well as tissue related morbidity (e.g. fibrosis). Whether the knowledge generated can translate into routine practical predictive measures, and/or more may give guidance for pathways which can serve as targets for biological interventional procedures is currently under investigation. It has in that aspect, however, become obvious that further elucidation of the clinical importance will demand access to large well characterized patient cohorts, and the establishment of these, and the associated biobanks are of outmost importance. A prime example is the ESTRO -GENEPI project involving thousands of patients. The presentation will attempt to give an overview and update of the biological basis of radiation related morbidity and its potential prediction, mainly using the genetic based variations in radiation related fibrosis as an example. 48 DNA DAMAGE RESPONSE IN RADIORESISTANT

GLIOMAS

S.C. Short 1, S. Bourne 1, C. Martindale ~, P. Johnston ~, S.P. Jackson 2. ~Gray Cancer Institute, Northwood UK, 2Gurdon Institute, University of Cambridge UK We have examined the response of radioresistant glioma ceils to radiation induced double strand breaks in detail to understand which aspects of the DNA damage response could be effective targets to produce selective radiosensitisation in these cells. M a t e r i a l a n d m e t h o d s : Human high grade glioma cell lines in which the radiation survival response has been described in detail previously were used to study the response to DNA damage at a cellular level in vitro. Phospho specific antibodies were used to identify signalling events following clinically relevant radiation doses. Immunofluorescent labelling was used to visualise repair protein foci, including H2AX phosphorylation, which was used to measure induction and the kinetics of repair of DNA double strand breaks. BrdUrd pulse labelling was used to measure cell cycle checkpoint responses in the same cells after doses between 0.2 and 2Gy. The same signalling events were also assessed in glioma cells pre-treated with other DNA damaging agents such as Temozolomide before irradiation. Results: Radiation induced double strand breaks could be measured directly using H2AX phosphorylation in human glioma cell lines. At 3 different time points after irradiation (0.5, 4 and 24h) H2AX phosphorylation appeared to be linearly related to dose in the clinically relevant dose range (0.2-2Gy). Repair kinetics assessed using the same assay suggested an exponential decay of unrepaired double strand breaks with time. As expected in p53 mutant cells there was no significant G1 checkpoint response following radiation. An effective, dose dependent G2 checkpoint response was evident. ATM dependent signalling to Chkl and Chk2 proteins occurred at the doses studied and is likely to be the relevant signalling mechanism. As has previously been described in glioma cells, we demonstrated a high level of Rad51 foci formation that also occurred in a dose dependent manner after radiation. These foci are thought to represent sites of homologous recombination (HR) that occurs at sites of collapsed replication forks in S-phase cells or following DNA double strand break induction in G2 when the homologous sister chromatid is available as a recombination partner. At low radiation doses in glioma cells, Rad51 foci formation was cell cycle dependent as measured by Cyclin A co-staining but at doses > l G y loci occurred in non G2 and S phase cells, Objective:

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March 12 - 15

Invited Speakers

suggesting that HR was occurring in other cell cycle phases. Pre-treatment with Temozolomide, which produces additional double strand breaks through futile mis-match repair and promotes G2 arrest, did not produce radiosensitisation, suggesting that double strand break repair in G2 is efficient in these cells. C o n c l u s i o n s : These data suggest that p53 mutated glioma cell lines may preferentially utilise G2 dependent repair mechanisms following clinically relevant doses of radiation. This implies that they will be sensitised by agents that specifically reduce DNA repair during G2. These include agents that target ATM dependent signalling to G2 checkpoint proteins or drugs that directly reduce the efficiency of Rad51 mediated homologous recombination. Early studies in which such agents have been used support this conclusion. 49 NOVEL CHEMO-RADIOTHERAPY TREATING H I G H - G R A D E G L I O M A

R.O. Mirimanoff Department of Radiation Oncology, Lausanne, Switzerland

APPROACHES

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CHUV, University of

Lessons from past studies: Until recently, post-operative radiotherapy (RT) was considered as standard therapy for highgrade glioma (HGG), whereas combined chemo-radiotherapy based on nitrosoureas showed only marginal advantage over RT alone. The reasons for the relative inefficiency of chemotherapy (CT) in HGG are multiple, and include tumor intrinsic resistance to CT, heterogeinity, genetic instability, poor intra-tumoral drug penetration, amongst other. Also most CT-RT schedules lacked a firm radiobiological rationale, and these studies had no translational research (TR) component. Lessons from the EORTC 2 6 9 8 1 / 2 2 9 8 1 / N C I C . C E 3 trial: This trial was the first to demonstrate that the addition of temozolomide (TMZ) to RT conferred a statistically significant and clinically meaningful survival advantage over RT alone in GBM. The concept of this trial is supported by demonstrated activity of TMZ combined to RT in preclinical studies using GBM models, on favourable pharmacokinetics andon a promising preliminary phase I-II study. Moreover a companion TR study to the EORTC/NCIC trial was undertaken to evaluate the MGMT promoter methylation status of the tumor in patients enrolled in the clinical trial. MGMT methylation, i.e. the silencing of the MGMT repair enzyme gene, was present in 45% of the cases and was associated not only with a better overall prognosis, but also with a superior response to the RT/TMZ combination. Novel approaches: Based on the EORTC/NCIC trial's results and its companion TR study, novel approaches are under way at EORTC in cooperation with other groups, a) One first avenue of research consists of integrating to RT/TMZ novel targeting agents for which there is a rationale for their use in GBM. Two inhibitors of angiogenesis, Celengitide and PTK/ZK 787 are currently tested, each in association with RT/TMZ in two phase I-II studies. The rationale and the trials will be presented, b) A second avenue consists of exploring intensified dose-dense TMZ with RT, taking into account the fact the continuous TMZ depletes intracellular MGMT. A large intergroup trial (RTOG/EORTC) will randomly assign patients to standard vs intensified TMZ with RT. In both approaches (a&b), related TR studies will be performed to identify specific markers or profiles related to response and prognosis. 5O I S TUMOR R A D I O S E N S I T I V I T Y PREDICTABLE?

A. Begg The Netherlands Cancer Institute, Amsterdam, The Netherlands The response of tumors to radiotherapy will depend on intrinsic cellular radiosensitivity and microenvironmental factors such as pH and hypoxia. In addition, tumor repopulation during radiotherapy will influence outcome for conventional (>5 weeks) treatment schedules. This in turn will depend on cell cycle checkpoints and the ability to respond to damage and cellular depletion. All these factors depend on which genes are expressed and/or mutated in individual tumors. At the present time, expression microarrays (transcript level) offer the most robust way of measuring expression profiles on a genome-wide scale. Neither mutations, nor protein expression, nor post-

translational modifications, nor protein function are measured with this technique. However, despite these limitations, cDNA/oligo microarrays have been shown to provide improved prognostic classification of several tumor types tumors over and above classical clinical parameters, and can predict the chance of metastasis in some studies. No large microarray studies have yet been published predicting outcome to a specific the[apy such as radiotherapy, although several studies addressing this problem are ongoing (including in the author's lab). The ability to predict normal tissue damage after radiotherapy may be possible (several conference proceeding reports suggest this), although whether this is possible/feasible from easily available cells/tissues (e.g. lymphocytes) remains to be seen, Proteomics (protein level) are an order of magnitude more complex both technically and in quantity (number of proteins versus number of transcripts) but theoretically offer greater promise ("proteins do the work"). "Simple" proteomics, e.g. immunohistochemistry (IHC), especially if coupled with tissue microarrays TMA, have already shown significant promise as predictors. The full potential of this IHC/TMA technique has yet to fully evaluated in the clinic, requiring the use of multiple markers (e.g. up to 30 would be feasible) and microarray-style analysis procedures, including cluster analyses, internal statistical validation procedures, etc. Such studies are expected to provide significantly improved predictors. At the DNA level, multiparameter FISH, comparative genomic hybridization and others also show promise but have some theoretical disadvantages and have not yet been evaluated sufficiently as predictors of outcome. Several single-gene studies or limited number (2-5) gene studies have already shown the ability to predict tumor response after radiotherapy. It is likely that assessment of several thousand genes will improve prediction, whether or not these are simply marker genes or genes actually involved in response. The increasing reliability (coupled with reduced costs) of measuring genome-wide gene expression, together with the increasing sophistication and application of bioinformatics approaches, makes this an attractive and promising route at present to better predict radiotherapy outcome. In summary, radiosensitivity should indeed be predictable, and significant progress is expected in this area within a few years. 51 DNA DAMAGE ASSAYS

E, Dikomey Laboratory of Radiobiology & Experimental Radiooncology, Clinic of Radiotherapy and Radiooncology; University Medical Center Hamburg-Eppendorf, Martinistrasse 52, 20246 Hamburg, Germany Objective: Tumour cell death mainly results from non- and mis-rejoined DNA damage that give rise to lethal chromosomal aberrations. Among the damage induced DNA double-strand breaks are considered to be the most relevant lesion leading to cell death. In this review we will concentrate on those techniques which can be especially used to measure the induction, repair and mis-repair of DNA double-strand breaks. Material and methods: Gel electrophoresis: For a long period gel electrophoresis applied either as a pulsed or a constant field was seen as the best technique to detect dsb. Due to the limited sensitivity other techniques are preferred these days. However, the graded field gel electrophoresis (GFGE) is still the best technique to measure the initial number of dsbs induced per cell and per Gy. This is of special relevance, because tumour cells were found grossly to vary in this number, which is one parameter that determines the cellular radiosensitivity. Southern hybridisation: Most of dsbs are rejoined correctly. However, there is a small fraction of non-correctly rejoined dsbs. These events can be detected by Southern hybridisation using rarely cutting restriction enzymes. The fraction on noncorrectly rejoined dsbs increases with increasing dose with substantial variation between tumour cell lines. Comet assay: This technique can be used to quantify dsbs in single cells. However, the dose range required is very high. In the clinics this assay was used to determine the fraction of hypoxic cells within a tumour, yH2AX foci: This technique can be used to detect dsb in a single cell even after a dose as low as 0.1 Gy and lower. It is possible to detect one single dsb in an adequate background. The technique has to be adapted to the specific