Keith A. Kunugi

The role of cell cycle redistribution in radiosensitization: Implications regarding the mechanism of fluorodeoxyuridine radiosensitization

PURPOSE Radiosensitization has previously been demonstrated in a human colon cancer cell line (HT-29) following a 2 h exposure to low, clinically relevant concentrations (0.05-0.5 microM) of fluorodeoxyuridine (FdUrd) (15). The sensitizer enhancement ratio value (measured at 10% survival) plateaued at approximately 1.7 between 16 and 32 h following removal of drug. Parallel studies investigating the effect of FdUrd on the distribution of cells throughout the cell cycle found that the percentage of cells in early S-phase increased to approximately 70% during the same period that maximal radiosensitization was noted. As a follow-up to these findings, experiments have been designed to investigate the contribution of this early S-phase delay to radiosensitization. METHODS AND MATERIALS Synchronized populations of HT-29 cells have been obtained with three separate techniques. Two involve the induction of a reversible metaphase arrest (with high pressure N2O or colcemid) followed by a shakeoff of mitotic cells. The third uses a plant amino acid, mimosine, to induce a reversible block at the G1/S boundary. Flow cytometry was used to analyze the degree of synchrony based on bromodeoxyuridine (BrdUrd) uptake and propidium iodide (PI) staining. Radiation survival curves were obtained on these synchronized populations to investigate changes in radiosensitivity through the cell cycle. Additionally, levels of thymidylate synthase (TS), the primary target of FdUrd cytotoxicity, were measured in each phase of the cell cycle using the TS 106 monoclonal antibody against human TS. RESULTS Synchronization with mitotic shakeoff produced relatively pure populations of cells in G1; however, the degree of synchrony in early S-phase was limited both by cells remaining in G1 and by cells progressing into late S-phase. These techniques failed to reveal increased radiosensitivity in early S-phase at 10% survival. An 18 h exposure to mimosine resulted in populations that more closely resembled the early S-phase enrichment following FdUrd exposure and revealed increased radiosensitivity during early S-phase. TS levels were noted to be only 1.3 times higher in S phase than in G0/G1. CONCLUSION Radiation survival data from cells synchronized with mitotic shakeoff techniques suggest that early S-phase delay is unlikely to be the primary mechanism of FdUrd radiosensitization. In contrast, the increased sensitivity seen in early S-phase with mimosine synchronized cells is similar to that seen with FdUrd. Although confounding biochemical pertubations cannot be ruled out, these data continue to suggest an association between early S-phase enrichment and radiosensitization. The significance of TS inhibition as a mechanism of FdUrd radiosensitization remains unclear.

Determination of exit skin dose for 192Ir intracavitary accelerated partial breast irradiation with thermoluminescent dosimeters.

PURPOSE Intracavitary accelerated partial breast irradiation (APBI) has become a popular treatment for early stage breast cancer in recent years due to its shortened course of treatment and simplified treatment planning compared to traditional external beam breast conservation therapy. However, the exit dose to the skin is a major concern and can be a limiting factor for these treatments. Most treatment planning systems (TPSs) currently used for high dose-rate (HDR) I192r brachytherapy overestimate the exit skin dose because they assume a homogeneous water medium and do not account for finite patient dimensions. The purpose of this work was to quantify the TPS overestimation of the exit skin dose for a group of patients and several phantom configurations. METHODS The TPS calculated skin dose for 59 HDR I192r APBI patients was compared to the skin dose measured with LiF:Mg,Ti thermoluminescent dosimeters (TLDs). Additionally, the TPS calculated dose was compared to the TLD measured dose and the Monte Carlo (MC) calculated dose for eight phantom configurations. Four of the phantom configurations simulated treatment conditions with no scattering material beyond the point of measurement and the other four configurations simulated the homogeneous scattering conditions assumed by the TPS. Since the calibration TLDs for this work were irradiated with C137s and the experimental irradiations were performed with I192r, experiments were performed to determine the intrinsic energy dependence of the TLDs. Correction factors that relate the dose at the point of measurement (center of TLD) to the dose at the point of interest (basal skin layer) were also determined and applied for each irradiation geometry. RESULTS The TLD intrinsic energy dependence for I192r relative to C137s was 1.041±1.78%. The TPS overestimated the exit skin dose by an average of 16% for the group of 59 patients studied, and by 9%-15% for the four phantom setups simulating treatment conditions. For the four phantom setups simulating the conditions assumed by the TPS, the TPS calculated dose agreed well with the TLD and MC results (within 3% and 1%, respectively). The inverse square geometry correction factor ranged from 1.023 to 1.042, and an additional correction factor of 0.978 was applied to account for the lack of charged particle equilibrium in the TLD and basal skin layer. CONCLUSIONS TPS calculations that assume a homogeneous water medium overestimate the exit skin dose for intracavitary APBI treatments. It is important to determine the actual skin dose received during intracavitary APBI to determine the skin dose-response relationship and establish dose limits for optimal skin sparing. This study has demonstrated that TLDs can measure the skin dose with an expanded uncertainty (k=2) of 5.6% when the proper corrections are applied.

Overexpression of the R2 subunit of ribonucleotide reductase in human nasopharyngeal cancer cells reduces radiosensitivity.

PURPOSERibonucleotide reductase is the rate-limiting enzyme in the de novo synthesis of deoxyribonucleotide triphosphates, which are utilized in both DNA synthesis and DNA repair. We reported previously that RR enzyme activity and R2 (catalytic subunit of RR) protein levels were increased after exposure to ionizing radiation (IR) in growth-arrested human tumor cells,1 suggesting that R2 protein expression regulates RR activity to allow for IR damage repair. Using isogenic human nasopharyngeal carcinoma cells in this study, we examine the relationship of overexpression of either the R1 regulatory subunit or the R2 catalytic subunit of RR to the cellular response of IR damage. MATERIALS AND METHODSWe used three isogenic human nasopharyngeal cancer cell lines previously derived by Zhou et al,2 including KB, the parental tumor cell line; KB/M1; an R1 protein-overexpressing clone stably transfected with human R1 complementary DNA; and KB/M2, a R2 protein-overexpressing clone stably transfected with human R2 complementary DNA. We initially characterized these isogenic human tumor cell lines in exponential growth for R2 protein expression, RR enzyme activity, and R2 protein changes during the cell cycle by flow cytometry. Subsequently, the IR response in these cell lines was determined by clonogenic survival, cell cycle changes occurring after IR, and an analysis of IR DNA damage determined by pulsed field gel electrophoresis. The effect of combining IR and hydroxyurea, a RR (R2) inhibitor, was also studied in KB and KB/M2 cells. RESULTSKB/M2 cells were found to have 4.5-fold higher R2 protein expression and a threefold higher RR enzyme activity in exponential growth than KB and KB/M1. Although R2 protein levels increased at the G1/S transition in all cell lines, KB/M2 cells also demonstrated consistently higher R2 protein levels throughout the cell cycle. Using a linear-quadratic analysis of IR clonogenic survival data, KB/M2 cells were more radioresistant than KB and KB/M1 cells, including both decreased α and decreased β values, a finding that correlates with increased reparable IR damage. KB/M2 cells also show a reduced G2 cell cycle arrest and fewer DNA double strand breaks 18 hours after IR (6 Gy). Exposure of KB/M2 cells to hydroxyurea (300 μM) after exposure to IR restored in vitro radiosensitivity in a manner similar to that found in KB and KB/M1 cells. DISCUSSIONAn increase in R2 protein levels and RR activity in KB/M2 cells results in IR resistance, which appears mediated by enhanced IR damage repair during G2. R1 protein overexpression in these isogenic human tumor cells (KB/M1) did not affect RR activity or IR response.

Global comparison of radiation and chemotherapy dose-response curves with a test for interaction

In the analysis of the effects of radiation or drugs on clonogenic survival data of mammalian cells, it is often advantageous to compare entire dose-response curves generated under different experimental conditions rather than to conduct single-dose comparisons. We propose a two-stage method for the global comparison of such curves. The first stage consists of individual fits of a flexible model to the dose-response sequences. The second stage treats the fitted coefficients as data and analyzes them jointly using a multivariate analysis of variance. For dose-response models of the type commonly used in the analysis of clonogenic survival data, this method allows the definition of a statistical test for interaction among treatments. That is, a test that the combination of agents produces a dose-response curve which is not what would be expected if the agents were acting multiplicatively (additively in the log cell survival scale). Analyses of cell survival curves by this method for experiments on iododeoxyuridine-mediated radiosensitization and on chemosensitization of bleomycin cytotoxicity in a human bladder cancer cell line (647V) are presented.

TH-E-AUD-04: Determination of Exit Skin Dose for MammoSite(R) RTS with TLD

Purpose: To accurately determine the exit skindose from MammoSite® Radiation Therapy System treatments using thermoluminescent dosimeters (TLDs) placed on the surface of the skin during each treatment fraction. Methods and Materials: Well‐characterized TLD‐100 chips were calibrated using 137 Cs to establish an energy response correction factor for 192 Ir measurements. TLD response vs. material thickness was measured and compared for thicknesses of Virtual Water™ and breast‐equivalent material from 2 to 10cm. Monte Carlo simulations were performed to determine the relationship between the dose to TLD and dose to basal skin layer for a range of treatment parameters (size and content of balloon, distance from skin, etc). Treatment planning system (TPS) predicted skindoses were also compared to the TLD measured skindose for 29 patients and two phantom setups. Results: The TLD‐100 energy response for 192 Ir relative to 137 Cs was determined to be 1.045. The TL vs. thickness curves for Virtual Water™ and breast‐equivalent material were found to be within 2.5% for all thicknesses studied for 192 Ir , and can be used interchangeably within the 2.5% limit, which falls within our measurement uncertainty of 3%. The Monte Carlo calculated dose to TLD agreed with the 1/r2 corrected basal skindose to within 0.5% for the entire range of treatment parameters studied when the dose to TLD vs. dose to water correction factor of 1.2 was applied. TPS predicted doses overestimated the TLD measured skindose by an average of 26% for the group of 29 patients studied, and by an average of 40% for the two phantom setups. Conclusions: TLDs placed on the surface of the breast during MammoSite RTS treatments can accurately measure the skindose when the proper correction factors are applied. The TLD measured dose is much more accurate than the TPS predicted dose.

A prototype, glassless densitometer traceable to primary optical standards for quantitative radiochromic film dosimetry

PURPOSE To evaluate a prototype densitometer traceable to primary optical standards and compare its performance to an EPSON Expression(®) 10000XL flatbed scanner (the Epson) for quantitative radiochromic film (RCF) dosimetry. METHODS A prototype traceable laser densitometry system (LDS) was developed to mitigate common film scanning artifacts, such as positional scan dependence and high noise in low-dose regions, by performing point-based measurements of RCF suspended in free-space using coherent light. The LDS and the Epson optical absorbance scales were calibrated up to 3 AU, using reference materials calibrated at a primary standards laboratory and a scanner calibration factor (SCF). Calibrated optical density (OD) was determined for 96 Gafchromic(®) EBT3 film segments before and after irradiation to one of 16 dose levels between 0 and 10 Gy, exposed to (60)Co in a polymethyl-methacrylate (PMMA) phantom. The sensitivity was determined at each dose level and at two rotationally orthogonal readout orientations to obtain the sensitometric response of each RCF dosimetry system. LDS rotational scanning dependence was measured at nine angles between 0°and 180°, due to the expected interference between coherent light and polarizing EBT3 material. The response curves were fit to the analytic functions predicted by two physical response models: the two-parameter single-hit model and the four-parameter percolation model. RESULTS The LDS and the Epson absorbance measurements were linear to primary optical standards to within 0.2% and 0.3% up to 2 and 1 AU, respectively. At higher densities, the LDS had an over-response (2.5% at 3 AU) and the Epson an under-response (3.1% and 9.8% at 2 and 3 AU, respectively). The LDS and the Epson SCF over the applicable range were 0.968% ± 0.2% and 1.561% ± 0.3%, respectively. The positional scan dependence was evaluated on each digitizer and shown to be mitigated on the LDS, as compared to the Epson. Maximum EBT3 rotational dependence was found to have a strong dependence on dose (0.1% and 34% at 30 mGy and 5 Gy, respectively). The preferred EBT3 polymerization axis angle was constant within experimental uncertainties. In its most sensitive orientation, the LDS-measured EBT3 sensitivity was 7.13 × 10(-4) ± 9.2 × 10(-6) AU/mGy, which represented a 4.5 fold increase over the Epson of 1.58 × 10(-4) ± 9.8 × 10(-6) AU/mGy. To first order approximations, EBT3 response was linear up to 500 mGy to within 0.80% and to within 7.5% for the most sensitive LDS and the Epson orientations, respectively. The corresponding single-hit and percolation model relative residual norms were 0.082 and 0.074 for LDS as compared to 0.29 and 0.18 for the Epson, which represented a significant increase in LDS-measured agreement with the simple physical model. Less sensitive LDS and the Epson orientations showed a marked decrease in the physical model agreement, which suggested that suboptimal readout device characteristics may be the origin of the complex sensitometric functional forms currently required for accurate RCF dosimetry. CONCLUSIONS The prototype densitometer was shown to be superior to a conventional scanner for quantitative RCF dosimetry based on physical models of film response. The Epson was shown to be a reliable tool for routine RCF dosimetry in a clinical setting, yet calibration to primary optical standards did not mitigate the necessity for complex, empirical functional form fitting.

PHOTON BEAM AUDITS FOR RADIATION THERAPY CLINICS: A PILOT MAILED DOSEMETER STUDY IN TURKEY

A thermoluminescent dosemeter (TLD) mailed dose audit programme was performed at five radiotherapy clinics in Turkey. The intercomparison was organised by the University of Wisconsin Radiation Calibration Laboratory (UWRCL), which was responsible for the technical aspects of the study including reference irradiations, distribution, collection and evaluation. The purpose of these audits was to perform an independent dosimetry check of the radiation beams using TLDs sent by mail. Acrylic holders, each with five TLD chips inside and instructions for their irradiation to specified absorbed dose to water of 2 Gy, were mailed to all participating clinics. TLD irradiations were performed with a 6 MV linear accelerator and (60)Co photon beams. The deviations from the TL readings of UWRCL were calculated. Discrepancies inside the limits of ±5 % between the participant-stated dose, and the TLD-measured dose were considered acceptable. One out of 10 beams checked was outside this limit, with a difference of 5.8 %.

SU-G-TeP3-02: Determination of Geometry-Specific Backscatter Factors for Radiobiology Studies

PURPOSE Radiation biology research relies on an accurate radiation dose delivered to the biological target. Large field irradiations in a cabinet irradiator may use the AAPM TG-61 protocol. This relies on an air-kerma measurement and conversion to absorbed dose to water (Dw) on the surface of a water phantom using provided backscatter factors. Cell or small animal studies differ significantly from this reference geometry. This study aims to determine the impact of the lack of full scatter conditions in four representative geometries that may be used in radiobiology studies. METHODS MCNP6 was used to model the Dw on the surface of a full scatter phantom in a validated orthovoltage x-ray reference beam. Dw in a cylindrical mouse, 100 mm Petri dish, 6-well and 96-well cell culture dishes was simulated and compared to this full scatter geometry. A reference dose rate was measured using the TG-61 protocol in a cabinet irradiator. This nominal dose rate was used to irradiate TLDs in each phantom to a given dose. Doses were obtained based on TLDs calibrated in a NIST-traceable beam. RESULTS Compared to the full scattering conditions, the simulated dose to water in the representative geometries were found to be underestimated by 12-26%. The discrepancy was smallest with the cylindrical mouse geometry, which most closely approximates adequate lateral- and backscatter. TLDs irradiated in the mouse and petri dish phantoms using the TG-61 determined dose rate showed similarly lower values of Dw. When corrected for this discrepancy, they agreed with the predicted Dw within 5%. CONCLUSION Using the TG-61 in-air protocol and given backscatter factors to determine a reference dose rate in a biological irradiator may not be appropriate given the difference in scattering conditions between irradiation and calibration. Without accounting for this, the dose rate is overestimated and is dependent on irradiation geometry.

SU-E-T-172: Characterization of TLD-100 (LiF:Mg,Ti) Microcube Energy Response in a Cylindrical Chamber Phantom

Purpose: To characterize the energy response of TLD-100 microcubes inside a Virtual Water chamber phantom. Methods: Four TLD microcubes were placed inside a water-proof Virtual Water (VW) chamber phantom and irradiated to a known dose on a Varian linac in a 1D water tank. These chamber phantoms were then replaced by TLD-100 chips inside a separate VW paddle and irradiated to the same dose. Each energy response reading was calculated as light output per unit dose in nC/cGy and normalized to a calibration set irradiated to the same dose in 60Co. The differences in response between the TLD chips and microcubes were then analyzed. Results: Across all energies, the average microcube response was less sensitive to energy than the average chip response with both falling consistently within 2.8% of previously established values in the literature Conclusion: TLD microcubes showed a lower average sensitivity to energy than their TLD chip counterparts. The use of TLD-100 microcubes inside the chamber phantom was validated against TLD-100 chips inside of VW paddles.

SU‐FF‐T‐391: A New Look at Helmet Output Factor Utility in Gamma Knife Treatments

Purpose: To characterize the volume dose distribution of Gamma Knife (GK) stereotactic radiosurgery units to compare point‐measurement based helmet factors and volume‐based measurements of dose.Method and Materials: Helmet factors were determined using radiochromic film and ionization chamber measurements as well as TLD arrays. Novel methods have been used to determine and characterize the volume of dose delivered by the GK unit. These methods include using large volume ionization chambers and deconvolving the measurement to remove volume averaging effects and creating a film phantom that allows us to map the entire dose volume in 3 dimensions by using cubes of radiochromic film. Results: The preliminary work done using traditional methods to determine helmet factors has shown that a single point measurement may be insufficient when delivering doses of the magnitude seen in GK treatments. Analysis using TLDs and radiochromic film indicate that non‐uniform dose distributions exist. It has also been shown that the isocenter of the GK does not always correspond with the point of maximum dose. Additionally, helmet factor determination is dependent on the size of the dosimeter being used, thus larger dosimeters demonstrate volume averaging effects preventing the proper determination of helmet factors. Conclusion: GK treatment is the preferred modality for a number of cranial treatments, and physicians are continually prescribing smaller margins. For this reason, it is vital that the dosimetry of the GK be representative of what the patient receives. While point and plane dosimetry techniques may be adequate when delivering radiation from one direction, 3‐D techniques must be developed for modalities like the GK, in which dose is delivered with multiple sources simultaneously and almost isotropically.