Differential effects of interstitial PDR and CLDR brachytherapy on cell cycle progression in a syngeneic rat prostate tumor model

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I. J. Radiation Oncology

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● Biology ● Physics

Volume 57, Number 2, Supplement, 2003

Stereotactic Hypofractionated High-Dose Irradiation for Patients with Stage I Non-Small Cell Lung Carcinoma: Clinical Outcomes in 241 Cases of a Japanese Multi-Institutional Study

H. Onishi,1 Y. Nagata,2 H. Shirato,3 K. Gomi,4 K. Karasawa,5 K. Hayakawa,6 Y. Takai,7 T. Kimura,8 A. Takeda,9 M. Hareyama,10 M. Kokubo,11 R. Hara12 1 Radiation Oncology, University of Yamanashi, Yamanashi, Japan, 2Radiation Oncology, Kyoto University, Kyoto, Japan, 3 Radiation Oncology, Hokkaido University, Sapporo, Japan, 4Radiation Oncology, Cancer Institute Hospital, Tokyo, Japan, 5 Radiation Oncology, Tokyo Metropolitan Komagome Hospital, Tokyo, Japan, 6Radiation Oncology, Kitasato University, Sagamihara, Japan, 7Radiation Oncology, Tohoku University, Sendai, Japan, 8Radiation Oncology, Hiroshima University, Hiroshima, Japan, 9Radiation Oncology, Tokyo Metropolitan Hiroo Hospital, Tokyo, Japan, 10Radiation Oncology, Sapporo Medical University, Sapporo, Japan, 11Radiation Oncology, Institute of Biomedical Research and Innovation, Kobe, Japan, 12Radiation Oncology, International Medical Center of Japan, Tokyo, Japan Purpose/Objective: Stereotactic irradiation (STI) for small lung tumors has been actively performed in Japan. The purpose of the current study is to retrospectively evaluate Japanese multi-institutional results of hypofractionated high-dose STI for patients with stage I non-small cell lung carcinoma (NSCLC). Materials/Methods: From April 1995 to October 2002, 241 patients (188 men, 53 women; age range 35–92, median 76 years) who had stage I (153 T1N0M0 and 88 T2N0M0) primary non-small cell (106 squamous cell, 102 adeno, and 33 others) lung carcinomas were treated with hypofractionated high-dose STI in 13 institutions. Of the 241 patients, 161 (67%) were medically inoperable due to mainly chronic pulmonary disease or high-age. Tumor size ranged from 7 to 58 mm with a median of 28 mm. The threedimensional treatment planning was performed using non-coplanar dynamic arcs or multiple static ports. Various techniques using the breathing control or gating method were applied in order to reduce respiratory internal margin. A total dose of 18–75 Gy at the isocenter in 1–22 fractions was irradiated within 20% of homogeneity in the planning target volume dose. The calculated biologic effective dose (BED(10)) at the isocenter using the linear-quadratic model ranged from 57 Gy to 180 Gy with a median of 108 Gy. Results: All patients completed the treatment with no complaints. During the follow up of 4 –72 months (median⫽18), radiation-induced pulmonary complications greater than NCI-CTC criteria grade 2 were noted in only 5 (2.1%) patients. Of 224 patients who were evaluated with CT, we observed CR and PR in 55 (22.7%) and 139 (62.1%) patients, respectively. The local recurrence occurred in 10.4% of total patients, and a higher local recurrence rate was observed (20.0% vs. 6.5%, p⫽0.04) when BED was ⬍ 100 Gy vs. ⬎ 100 Gy. Regional lymphnodes and distant metastasis occurred in 5.8% and 12.4% of total patients, respectively. The intercurrent death was observed in 29 (12.0%), mostly in inoperable patients. The 3-year overall and cause-specific survival rates were 56.0% and 71.8%, respectively. The 3-year cause-specific survival rate of stage IA and IB patients were 75.8% and 62.9%, respectively. The 3-year cause-specific survival rates were significantly high (78.7% vs. 58.6%, p⫽0.006) when BED was ⬎ 100 Gy vs. ⬍ 100 Gy. Conclusions: Hypofractionated high-dose STI is a feasible and beneficial method for the curative treatment of patients with stage I NSCLC. A considerable number of intercurrent deaths were observed in inoperable patients. The local control and survival rate was high when BED was ⬎ 100 Gy. Based on these good clinical outcomes, we will soon start a multi-institutional phase II study of hypofractionated high-dose (48Gy / 4 fractions; BED⫽105.6Gy) STI for patients with stage I NSCLC.

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Differential Effects of Interstitial PDR and CLDR Brachytherapy on Cell Cycle Progression in a Syngeneic Rat Prostate Tumor Model

W. Harms,1 P. Peschke, V. Ehemann,3 K. Weber,1 I. Zuna, J. Debus,1 M. Wannenmacher1 1 Clinical Radiology, University of Heidelberg, Heidelberg, Germany, 2Radiation Oncology, German Cancer Research Center, Heidelberg, Germany 3Pathology, University of Heidelberg, Heidelberg, Germany Purpose/Objective: Radiobiological models based on incomplete repair calculations (linear quadratic formalism) suggest that PDR (pulsed dose rate) should have under certain conditions the same radiobiological effectiveness as CLDR (continuous low dose rate) brachytherapy. Possible differential effects of these two fractionation schedules on cell cycle progression are not considered in these models, but may play an important role. The aim of this study was therefore: 1) to establish reproducible flow cytometrical measurements in solid tumors, 2) to evaluate the effects of interstitial CLDR and PDR brachytherapy on cell cycle distribution in a syngeneic rat prostate tumor model. Materials/Methods: Interstitial PDR and CLDR brachytherapy were administered to Dunning prostate R3327-AT1 carcinomas transplanted subcutaneously into the right thigh of Copenhagen rats. Doses of 20 and 40 Gy were administered in each study arm (CLDR versus PDR). Interstitial PDR was carried out using a 37 GBq 192-Ir source with 0.75 Gy/pulse and hour. CLDR was administered with a centrally implanted seed with a dose rate of 0.75 Gy/h. The dose was prescribed to the tumor surface (5 mm source distance, tumor diameter 10 mm). Endpoint of this study was the cell cycle distribution of the diploid and aneuploid cells at 4, 24, 48, 72, 96, and 120 h after the initiation of brachytherapy. Tumors either implemented with an empty tubing system (n⫽5) or under undisturbed growth (n⫽5) served as controls. Three animals were irradiated per time point and exposure condition. At least two flow cytometrical analyses were carried out per animal. Flow cytometry was performed after 6-diamidino-2-phenyl-indole (DAPI) staining of disintegrated tumor tissue fragments using a PAS II flow cytometer (Partec, Germany). For cell cycle analysis and ploidy status the resultant histograms covering 30000 –90000 cells were evaluated by the Multicycle program (Phoenix Flow Systems, USA). Results: By flow cytometric analysis this tumor consisted of a mixture of diploid and aneuploid cells, with the latter cohort possessing a constant DNA-Index of 1.9⫾0.06. Only the aneuploid cell fraction, which mainly represented the tumor population, was used for further analysis. Comparison of sham treated (empty tube) and untreated controls did not show a differential effect of tube insertion on cell cycle regulation. Under both treatment modalities (dose 20 Gy), the fraction of aneuploid cells in G2M-phase increased significantly within 48 h (PDR, Mann-Whitney Test p⬍0.05) and 24 h (CLDR, Mann-Whitney Test p⬍0.05) after initiation of therapy (see Table, G2M values are given as mean percentages of the aneuploid fraction, dose 20 Gy). To avoid multiple statistical testing at the different time points the Kolmogorov-Smirnov was used as a non-parametric statistical test to compare the corresponding cell cycle phases of the two treatment arms (e.g. G1 CLDR vs G1 PDR). After irradiation with 20 Gy of cell cycle progression was significantly different for PDR and CLDR with regard to

Proceedings of the 45th Annual ASTRO Meeting

all cell cycle phases (G1: p⬍0.05; S: p⬍0.05; G2M: p⬍0.05). These differences diminished after irradiation with 40 Gy where only the G1 phase showed a borderline significance (p⫽0.0474). Conclusions: PDR and CLDR brachytherapy displayed a statistically significant effect on cell cycle progression. Cell cycle progression represents another important factor beneath incomplete repair when comparing the radiobiological effects of different fractionation schedules. These data further expand our mechanistical understanding of PDR and CLDR brachytherapy.

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Recognition of Oxidative DNA Lesions by the DNA Mismatch Repair Pathway

P. Hungspreugs,1 T.M. Sofinowski,2 L.A. Uzdilla,1 S.M. Matthews,1 D.M. Wilson,2 T.M. Wilson1 1 Radiation Oncology, University of Maryland, Baltimore, MD, 2Laboratory of Molecular Gerontology, National Institute of Aging, Baltimore, MD Purpose/Objective: The goal of this study was to characterize the role of the DNA mismatch repair (MMR) pathway in the recognition and repair of oxidative DNA damage. Oxidative DNA damage represents a major threat to genomic stability and includes single-strand breaks as well as base damage and modifications. Sources of reactive oxygen species (ROS) include chronic inflammation, cellular metabolism, and a variety of environmental factors including chemical mutagens and ionizing radiation. Inactivation of the MMR pathway by mutation or epigenetic processes results in microsatellite instability (MSI) and has been correlated with a variety of sporadic cancers as well as the inherited cancer syndrome Hereditary Non-Polyposis Colorectal Cancer (HNPCC). In addition to correcting replication errors, a role for MMR in sensing oxidative DNA damage and signaling for cell death has been established as mouse embryonic stem cells deficient in MMR are more resistant to low level g-irradiation (1). The increased survival exhibited by the ES cells was attributed to a failure to efficiently execute programmed cell death (apoptosis) in response to radiation exposure. Materials/Methods: Gel mobility shift assays were performed using 32P end-labeled 42-mer DNA duplexes possessing a central ⫹CA extrahelical loop, single-strand DNA break, abasic site, deoxyribose phosphate lesion, or a single nucleotide gap. Purified MMR lesion recognition complexes as well as the DNA BER polymerase, Pol beta, were incubated with the lesions and separated on an 5% polyacrylamide gel followed by visualization on a BioRad Molecular Imager phosphoimager. Primer extension assays were performed using radiolabeled nucleotides and a 34-mer DNA duplex possessing a single nucleotide gap. The extension products were separated on an 18% sequencing gel and visualized on a phosphoimager. Results: We have determined that the MMR lesion recognition complex specifically recognizes and interacts with single-strand breaks as well as intermediates of the base excision repair (BER) pathway including abasic sites, deoxyribose phosphate lesions, and single nucleotide gaps. The affinity of the MMR complex for these lesions, as determined by measuring dissociation constants, was found to be equal to that of the MMR complex for its ideal mispair substrate. Primer extension assays demonstrated a 4- to 6-fold enhanced level of nucleotide incorporation by polymerase beta in the presence of the MMR complex. Conclusions: The results of our studies demonstrate that the MMR pathway is capable of recognizing oxidative DNA lesions and functionally interacting with the BER pathway. The recognition of these lesions by the MMR pathway provides a basis for a mismatch-repair-dependent apoptosis pathway that contributes to ROS induced cytotoxicity. The resistance of MMR deficient cells to cell killing by oxidative DNA damage may play a role in the increased cancer risk characteristic of HNPCC. 1. DeWeese TL, Shipman JM, Larrier NA, Buckley NM, Kidd LR, Groopman JD, Cutler RG, Riele H, Nelson WG. Mouse embryonic stem cells carrying one or two defective Msh2 alleles respond abnormally to oxidative stress inflicted by low-level radiation. Proc Natl Acad Sci USA 1998;95:11915–20.

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Decreased Expression of the DNA Mismatch Repair Gene, Mlh1, Under Hypoxic Stress in Mammalian Cells

P.M. Glazer Therapeuctic Radiology, Yale University, New Haven, CT Purpose/Objective: The hypoxic tumor microenvironment has been shown to contribute to genetic instability and to promote the malignant phenotype of solid tumors. As one possible mechanism for this effect, we investigated the function of selected DNA repair pathways under conditions of hypoxic cell stress. Materials/Methods: A series of human and mouse cell lines were exposed to defined cell culture conditions, including normoxia and hypoxia. In some cases, cell cultures were supplemented with agents designed to mimic hypoxia (such as desferrioxamine) or to alter gene regulation (such as azacytidine or trichostatin A). Cells were harvested at selected time points for Western and Northern blot analyses of repair gene expression. Genetic instability under the selected culture conditions was also measured using mutation reporter gene assays. Results: Expression of the DNA mismatch repair (MMR) gene, Mlh1, was specifically reduced under hypoxia, whereas expression of other MMR genes, including Msh2, Msh6, and Pms2, was not altered at the mRNA level. However, levels of the PMS2 protein were reduced, consistent with destabilization of PMS2 in the absence of its heterodimer partner, MLH1. The hypoxia-induced reduction in Mlh1 mRNA was prevented by the histone deacetylase inhibitor, trichostatin A, suggesting that

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