$312 Friday/Saturday, 20-21 September 2002 Posters

1071 Poster effective means of delivering highly conformal radiation therapy. By increas- Pro l i fe ra t ion markers Ki67 and BrdU in GLF squamous cel l ing the conformality around the targeted tumor, the therapeutic ratio (tumor carcinoma xenografts during fractionated irradiation dose:normal tissue dose) is substantially enhanced, thereby leading to W. Eicheler, F. Hesse/, M. Baumann decreased toxicity. However, the time required to administer each daily Medical Faculty, TU Dresden, Department of Radiation Oncology, Dres- treatment may be substantially longer than with conventional treatment. The den, Germany radiobiological consequences of this prolongation have not been fully exam-

ined. We calculated the average radiation dose-rates for IMRT and com- Proliferation of clonogenic tumor cells during fractionated irradiation is a pared these to the dose-rates used in clinical studies specifically designed main problem in radiotherapy, leading to loss of local tumor control with to exploit the different radiobiology of low-dose rate and "pulsed low dose- increased overall treatment time. The amount and possible changes of rate" (PLDR) radiotherapy. (1) hypoxic tumor cells may also influence the outcome of radiation therapy. Materials/Methods: Fifteen patients in our clinic were treated using IMRT To get more insight into the biological basis of this effect, we irradiated GLF over the past 6 months. The time required to treat each patient was record- human squamous cell carcinoma grown in nude mice with 18 fractions with- ed. Twenty five other patients were treated according to a treatment in overall treatment times of 18, 36, or 54 days. Irradiation was given either schema specifically designed to administer pulsed low-dose rate radiother- under ambient or homogeneous hypoxic (clamped) conditions. Fraction apy (aiming to deliver approximately 6 cGy/min). The time required for treat- sizes were 2 Gy in the ambient and 3 Gy in the clamped tumors, assuming ment and the consequent dose-rates of each cohort were compared. Treat- an oxygen enhancement ratio of 2,7. Tumor cell counts and the proliferation ment time and dose-rates of conventional radiotherapy were also markers Ki67 and BrdU labelling indices (LI) were determined in randomly evaluated. selected tumor areas after 0, 6, 12 and 18 fractions. Results: The average time required to treat patients using IMRT was 25 We observed a significant dose dependent decrease in the tumor cell num- minutes (range 15-35 rain). This amounted to an a v~. rage dose-rate of 7.2 ber in the ambient and the clamped tumors. The effect was most pro- cGy/min with a range as low as 5.1 cGy/min. This ~as comparable to the nounced in the tumors after daily irradiation. In the clamped treatment arms, PLDR treatment rates which averaged 6 cGy/min. Conventional treatment the Ki67 LI dropped significanty during daily and bidaily irradiation, but not rates typically ranged from 30-300 cGy min. in the tridaily treatment arm. Under ambient conditions, the Ki67 LI did not Conclusions: The calculated radiation dose-rates for patients treated with change in either irradiaton schedule. The same effects were seen with the IMRT were substantially lower than in conventional radiation therapy and S-phase marker BrdU Lt. were comparable to the dose-rates used in our pulsed low dose-rate clini- A clearcut time factor is seen in tumors after clamped as welt as ambient cal investigations. Although IMRT improves tumor conformality, thereby irradiation. The decrease in the proliferaton markers Ki67 and BrdU in GLF enhancing the therapeutic ratio and decreasing complications, the lower tumors corresponds with the overall treatment time of a fractionated radio- dose-rate, with its potential consequences on tumor control should be con- therapy, sidereal and further investigated.

Supported by the Deutsche Forschungsgemeinschaff (BA1433) 1. Welsh J, Howard S. Fractionated Low Dose-Rate Whole Abdominal Irra- diation For Recurrent Malignancy. The Cancer Journal 7,547-8; 2001

1072 Poster Relationship between DNA-PK act iv i ty , in t r ins ic rad ia t ion 1074 Poster sensitivity, and radiation-induced chromosome aberrations Glioblastoma cell line killing by fractionated radiation vio- in human tumor cell lines lares the equal effect per fraction assumption

M. Sarvi, L. Sappe/sa, E. B/azek M. Rave-Frank 1, T. Martin 1, P. Virsik-KSpp 2, H. Schmidberger 1 Rush-Presbyterian-St. Luke's Medica/ Center, Radiation Onco/ogy, Chica- 1University GSffingen, Radiotherapy and Radiation Onco/ogy, Gdttin- go, U.S.A. gen, Germany 2Univereity G6ttingen, Clinica/ Radiation Bio/ogy and Physics, Gdttin- Introduction: Cell survival after fractionated irradiation, the effective surviv- gen, Germany ing fraction (ESF), should be the best in vitro predictor of tumor control.

When n fractions (fx) of dose d are separated sufficiently for complete repair Purpose: DNA-dependent protein kinase (DNA-PK) is a complex of DNA- of sublethal damage, equal effect per fx predicts: ESFLQ(n x d) = [exp(-ad PK catalytic subunit and the DNA end-binding Ku70/Ku86 heterodimer. This - bd2)]n (Eq. 1) We directly measured ESF for two glioblastoma multiforme complex is required for DNA double strand break repair by nonhomologous (GBM) cell lines, U87MG and T98G, for comparison with ESFLQ predicted end joining and therefore plays an essential role in the cellular response to by Eq. 1 using a and b values from single-fx survival curves. ionising radiation. We analysed the relationship between DNA-PK activity, Materials and Methods: Single-cell suspensions were serially diluted into intrinsic radiation sensitivity, and yields of radiation-induced excess acentric 96-well plates at appropriate densities for each dose and irradiated 4 h later. fragments in six human tumor cell lines. Fractionated irradiations were performed daily: 1-30 fx of 2 Gy/d, 1-15 fx of Material and methods: Cells representing HNSCC (GR-145, ZMK-1), 4 Gy/d, or 1-10 fx of 6 Gy/d. Plates were stained 4 wk after the last fx. WCF glioma (MO59K, MO59J), lung (A549) and breast (MCF7) cancer were irra- is the fraction of wells without colonies of at least 50 cells. The plating effi- diated with single doses of 200 kV X-rays. Radiosensitivity was determined ciency PE = [-In (WCF)]/N, where N is the average number of cells plat- by a clonogenic assay. Genomic yields of excess acentric fragments, an ed/well. The surviving fraction SF(n x d) = [-In (WCF)]/(N*PE). Single-frac- indirect measure of unrepaired DNA damage, were scored in Giemsa- tion survivals were fitted by nonlinear regression to the LQ model: SF(d) = stained metaphases. DNA-PK was isolated according to Lees-Miller et al., exp (-ad - bd2), and the expected ESFLQ was calculated using Eq. 1. and activity was determined using the SigmaTECT DNA-Dependent Protein Results: The LQ parameters for U87MG were a = 0.038 ± 0.004; b = 0.007 Kinase Assay System. + 0.002, while for T98G, a = 0.050 ± 0.007; b = 0.018 ± 0.003 (means ± Results: Within the cell lines tested, GR-145 and MO59K were the most S.E.). ESF(2 Gy x,~-8) = 0,058 ± 0.013 for U87MG and 0.0046 ± 0.0019 radioresistant and had the highest DNA-PK activity. A549, ZMK-1 and MCF- for T98G. Scoring of 48- and 60-Gy plates was complicated by giant cells 7 showed an intermediate radiosensitivity and an intermediate DNA-PK with ambiguous morphologies. activity. MO59J, a DNA-PK deficient cell line was very radiosensitive. In Table 1. Ratio of Observed to Predicted ESF [ESF/ESFLQ] general, cell lines with low DNA-PK-activity had higher yields of radiation- Total 87MG (dose/fx) T98G (dose/fx)

Dose 2 4 6 2 .4 6 induced chromosome aberrations, especially of excess acentric fragments 12 22.4 12.5 9.6 0.87 0.36 0.35 as compared to cells with high DNA-PK-activity. 24 805 285 122 0.39 0.14 0.062 Conclusion: In the tumor cell lines tested, high intrinsic radiosensitivity was 36 25700 3996 951 0.10 0.003 0.002 correlated with relatively low DNA-PK activity, as measured by the Sigma- Conclusions: For U87MG and T98G, the LQ model and the equal.effect TECT assay, and increased levels of unrepaired DNA damage manifested assumption are inaccurate predictors of ESF. U87 is more resistant, while as high yields of radiation-induced excess acentric fragments. T98G is less resistant, than predicted by Eq. 1. Only for T98G cells

exposed to 6 fx of 2 Gy do the predicted and experimental values agree. 1073 Poster Repopulation, not considered in Eq. t , could partially explain our observa- Radiat ion dose-rate considerations with intensity modulated tions for U87MG but not for T98G. Factors such as cell cycle reassortment, radiation therapy dose-dependent SLDR kinetics, gene induction/repression by radiation, J. Welsh, J. Fowler, M. Burkhamer, M. Mehta, S. Howard and/or inherent heterogeneity might contribute to the large observed dis- University of Wisconsin, Human Oncology crepancies. Our results challenge the assumption of equal effect per radi-

ation fraction, at least for these GBM cell lines in monolayer culture. Intensity modulated radiation therapy (IMRT) has become an important and