Fitting tumor control probability models to biopsy outcome after three-dimensional conformal radiation therapy of prostate cancer: pitfalls in deducing radiobiologic parameters for tumors from clinical data.

PURPOSE: The goal of tumor control probability (TCP) models is to predict local control for inhomogeneous dose distributions. All existing fits of TCP models to clinical data have utilized summaries of dose distributions (e.g., prescription dose). Ideally, model fits should be based on dose distributions in the tumor, but usually only dose-volume histograms (DVH) of the planning target volume (PTV) are available. We fit TCP models to biopsy outcome after three-dimensional conformal radiation therapy of prostate cancer using either a dose distribution summary or the full DVH in the PTV. We discuss differences in the radiobiologic parameters and dose-response curves and demonstrate pitfalls in interpreting the results. METHODS AND MATERIAL: Two mechanistic TCP models were fit with a maximum likelihood technique to biopsy outcome from 103 prostate patients treated at Memorial Sloan-Kettering Cancer Center. Fits were performed separately for different patient subgroups defined by tumor-related prognostic factors. Fits were based both on full DVHs, denoted TCP(DVH(calc)), and, alternatively, assuming a homogeneous PTV dose given by the mean dose (Dmean) of each DVH, denoted TCP(Dmean(calc)). Dose distributions for these patients were very homogeneous with any cold spots located on the periphery of the PTV. These cold spots were uncorrelated with biopsy outcome, likely because the low-dose regions may not contain tumor cells. Therefore, fits of TCP models that are potentially sensitive to cold spots (e.g., TCP(DVH(calc))) likely give biologic parameters that diminish this sensitivity. In light of this, we examined differences in fitted clonogenic cell number, N(C), or density, rho(C), surviving fraction after 2 Gy, SF(2), or radiosensitivity, alpha, and their standard deviations in the population, sigma(SF(2)) and sigma(alpha), resulting from fits based on TCP(DVH(calc)) and TCP(Dmean(calc)). Dose-response curves for homogeneous irradiation (characterized by TCD(50), the dose for a TCP of 50%) and differences in TCP predictions calculated from the DVH using alternatively derived parameters were evaluated. RESULTS: Fits of TCP(Dmean(calc)) are better (i.e., have larger likelihood) than fits of TCP(DVH(calc)). For TCP(Dmean(calc)) fits, matching values of SF(2) and sigma(SF(2)) (or alpha and sigma(alpha)) exist for all N(C) (rho(C)) above a threshold that give fits of equal quality, with no maximum in likelihood. In contrast, TCP(DVH(calc)) fits have maximum likelihood for high SF(2) (low alpha) values that minimize effects of cold spots. Consequently, small N(C) (rho(C)) values are obtained to match the observed control rate. For example, for patients in low-, intermediate-, and high-risk groups, optimum values of SF(2) and N(C) are 0.771 and 3.3 x 10(3), 0.736 and 2.2 x 10(4), and 0.776 and 1.0 x 10(4), respectively. The TCD(50) of dose-response curves for intermediate-risk patients is 2.6 Gy lower using TCP(DVH(calc)) parameters (TCD(50) = 67.8 Gy) than for TCP(Dmean(calc)) parameters (TCD(50) = 70.4 Gy). TCP predictions calculated from the DVH using risk group-dependent TCP(Dmean(calc)) parameters are up to 53% lower than corresponding calculations with TCP(DVH(calc)) parameters. CONCLUSION: For our data, TCP parameters derived from DVHs likely do not reflect true radiobiologic parameters in the tumor, but are a consequence of the reduced importance of low-dose regions at the periphery of the PTV. Deriving radiobiologic parameters from TCP(Dmean(calc)) fits is not possible unless one parameter is already known. TCP predictions using TCP(DVH(calc)) and TCP(Dmean(calc)) parameters may differ substantially, requiring consistency in the derivation and application of model parameters. The proper derivation of radiobiologic parameters from clinical data requires both substantial dose inhomogeneities and understanding of how these coincide with tumor location.

Late rectal bleeding after conformal radiotherapy of prostate cancer. II. Volume effects and dose-volume histograms.

PURPOSE AND OBJECTIVE: Late rectal bleeding is a potentially dose limiting complication of three-dimensional conformal radiotherapy (3D-CRT) for prostate cancer. The frequency of late rectal bleeding has been shown to increase as the prescription dose rises above 70 Gy. The purpose of this study is to identify features of the cumulative dose-volume histogram (DVH) for the rectal wall that correlate with late rectal bleeding after 3D-CRT for prostate cancer. METHODS AND MATERIALS: Follow-up information on rectal bleeding is available for 261 and 315 patients treated using 3D-CRT at Memorial Sloan-Kettering Cancer Center for Stage T1c-T3 prostate cancer with minimum target doses of 70.2 and 75.6 Gy, respectively. All patients in this study were treated with a coplanar 6-field technique (2 lateral and 4 oblique fields). Patients were classified as having rectal bleeding if they bled (> or = Grade 2) before 30 months, and nonbleeding (< or = Grade 1) if they were without bleeding at 30 months, using the RTOG morbidity scale. Rectal bleeding was observed in 13 and 38 of the patients treated at 70.2 and 75.6 Gy, respectively. Treatment plans were analyzed for 39 nonbleeding and 13 bleeding patients receiving 70.2 Gy, and 83 nonbleeding and 36 bleeding patients receiving 75.6 Gy. Dose-volume histograms (DVHs) for the anatomic rectal wall were calculated. Average DVHs of the bleeding and nonbleeding patients were generated, and a permutation test was used to assess the significance of differences between them, for each dose group. The confounding effect of total rectal wall volume (V(RW)) was removed by calculating the average differences in DVHs between all combinations of bleeding and nonbleeding patients with similar V(RW)s. Finally, multivariate analysis using logistic regression was performed to test the significance of the DVH variables in the presence of anatomic, geometric, and medical variables previously found to correlate with rectal bleeding in a companion analysis of the same patients. RESULTS: The area under the average percent volume DVH for the rectal wall of patients with bleeding was significantly higher than those of patients without bleeding in both dose groups (p = 0.02, 70.2 Gy; p < 0.0001, 75.6 Gy). However, small V(RW)s were associated with rectal bleeding (p = 0.06, 70.2 Gy; p < 0.01, 75.6 Gy), resulting in an increase in average percent volumes exposed to all doses for patients with rectal bleeding. For patients with similar V(RW)s, rectal bleeding was significantly correlated with the volumes exposed to 46 Gy in both dose groups (p = 0.02, 70.2 Gy; p = 0.005, 75.6 Gy, tolerance in V(RW): 5 ccs). For the 75.6 Gy dose group, the percent volume receiving 77 Gy was significantly correlated with rectal bleeding (p < 0.005). Bivariate analysis using logistic regression, including V(RW) together with a single DVH variable, showed good agreement with the above analysis. Multivariate analysis revealed a borderline significant correlation of the percent volume receiving 71 Gy in the 70.2 Gy dose group. It also showed that the DVH variables were highly correlated with geometric and dosimetric variables previously found to correlate with rectal bleeding in multivariate analysis. CONCLUSION: Significant volume effects were found in the probability of late rectal bleeding for patients undergoing 3D-CRT for prostate cancer with prescription doses of 70.2 and 75.6 Gy. The percent volumes exposed to 71 and 77 Gy in the 70.2 and 75.6 Gy dose groups respectively were significantly correlated with rectal bleeding. The independent correlation of small V(RW) with rectal bleeding may indicate the existence of a functional reserve for the rectum. The independent association with larger percent volumes exposed to intermediate doses ( approximately 46 Gy) seen in both dose groups may indicate that a large surrounding region of intermediate dose may interfere with the ability to repair the effects of a central high dose region.

Analysis of biopsy outcome after three-dimensional conformal radiation therapy of prostate cancer using dose-distribution variables and tumor control probability models.

PURPOSE: To investigate tumor control following three-dimensional conformal radiation therapy (3D-CRT) of prostate cancer and to identify dose-distribution variables that correlate with local control assessed through posttreatment prostate biopsies. METHODS AND MATERIAL: Data from 132 patients, treated at Memorial Sloan-Kettering Cancer Center (MSKCC), who had a prostate biopsy 2.5 years or more after 3D-CRT for T1c-T3 prostate cancer with prescription doses of 64.8-81 Gy were analyzed. Variables derived from the dose distribution in the PTV included: minimum dose (Dmin), maximum dose (Dmax), mean dose (Dmean), dose to n% of the PTV (Dn), where n = 1%,...,99%. The concept of the equivalent uniform dose (EUD) was evaluated for different values of the surviving fraction at 2 Gy (SF(2)). Four tumor control probability (TCP) models (one phenomenologic model using a logistic function and three Poisson cell kill models) were investigated using two sets of input parameters, one for low and one for high T-stage tumors. Application of both sets to all patients was also investigated. In addition, several tumor-related prognostic variables were examined (including T-stage, Gleason score). Univariate and multivariate logistic regression analyses were performed. The ability of the logistic regression models (univariate and multivariate) to predict the biopsy result correctly was tested by performing cross-validation analyses and evaluating the results in terms of receiver operating characteristic (ROC) curves. RESULTS: In univariate analysis, prescription dose (Dprescr), Dmax, Dmean, dose to n% of the PTV with n of 70% or less correlate with outcome (p < 0.01). The area under the ROC curve for Dmean is 0.64. In contrast, Dmin (p = 0.6), D98 (p = 0.2) or D95 (p = 0.1) are not significantly correlated with outcome. The results for EUD depend on the input parameter SF(2): EUD correlates significantly with outcome for SF(2) of 0.4 or more, but not for lower SF(2) values. Using either of the two input parameters sets, all TCP models correlate with outcome (p < 0.05; ROC areas 0.60-0.62). Using T-stage dependent input parameters, the correlation is improved (logistic function: p < 0.01, ROC area 0.67, Poisson models: p < 0.01, ROC areas 0.64-0.66). In comparison, the ROC area is 0.68 for the combination of Dmean and T-stage. After multivariate analysis, a model based on TCP, D20 and Gleason score is the best overall model (ROC area 0.73). However, an alternative model based on Dmean, Gleason score, and T-stage is competitive (ROC area 0.70). CONCLUSION: Biopsy outcome after 3D-CRT of prostate cancer at MSKCC is not correlated with Dmin in the PTV and appears to be insensitive to cold spots in the dose distribution. This observation likely reflects the fact that much of the PTV, especially at the periphery, may not contain viable tumor cells and that the treatment margins were sufficiently large. Therefore, the predictive power of all variables which are sensitive to cold spots, like TCPs with Poisson models and EUD for low SF(2), is limited because the low dose region may not coincide with the tumor location. Instead, for MSKCC prostate cancer patients with their standardized CTV definition, substantial target motion and small dose inhomogeneities, Dmean (or any variable that downplays the effect of cold spots) is a very good predictor of biopsy outcome. While our findings may indicate a general problem in the application of current TCP models to clinical data, these conclusions should not be extrapolated to other disease sites without careful analysis.

Late rectal toxicity after conformal radiotherapy of prostate cancer (I): multivariate analysis and dose-response.

PURPOSE: The purpose of this paper is to use the outcome of a dose escalation protocol for three-dimensional conformal radiation therapy (3D-CRT) of prostate cancer to study the dose-response for late rectal toxicity and to identify anatomic, dosimetric, and clinical factors that correlate with late rectal bleeding in multivariate analysis. METHODS AND MATERIALS: Seven hundred forty-three patients with T1c-T3 prostate cancer were treated with 3D-CRT with prescribed doses of 64.8 to 81.0 Gy. The 5-year actuarial rate of late rectal toxicity was assessed using Kaplan-Meier statistics. A retrospective dosimetric analysis was performed for patients treated to 70.2 Gy (52 patients) or 75.6 Gy (119 patients) who either exhibited late rectal bleeding (RTOG Grade 2/3) within 30 months after treatment (i.e., 70.2 Gy-13 patients, 75. 6 Gy-36 patients) or were nonbleeding for at least 30 months (i.e., 70.2 Gy-39 patients, 75.6 Gy-83 patients). Univariate and multivariate logistic regression was performed to correlate late rectal bleeding with several anatomic, dosimetric, and clinical variables. RESULTS: A dose response for >/= Grade 2 late rectal toxicity was observed. By multivariate analysis, the following factors were significantly correlated with >/= Grade 2 late rectal bleeding for patients prescribed 70.2 Gy: 1) enclosure of the outer rectal contour by the 50% isodose on the isocenter slice (i.e., Iso50) (p < 0.02), and 2) smaller anatomically defined rectal wall volume (p < 0.05). After 75.6 Gy, the following factors were significant: 1) smaller anatomically defined rectal wall volume (p < 0.01), 2) higher rectal D(max) (p < 0.01), 3) enclosure of rectal contour by Iso50 (p < 0.01), 4) patient age (p = 0.02), and 5) history of diabetes mellitus (p = 0.04). In addition to these five factors, acute rectal toxicity was also significantly correlated (p = 0.05) with late rectal bleeding when patients from both dose groups were combined in multivariate analysis. CONCLUSION: A multivariate logistic regression model is presented which describes the probability of developing late rectal bleeding after conformal irradiation of prostate cancer. Late rectal bleeding correlated with factors which may indicate that a greater fractional volume of rectal wall was exposed to high dose, such as smaller rectal wall volume, inclusion of the rectum within the 50% isodose on the isocenter slice, and higher rectal D(max).

Changes in histology and fibrogenic cytokines in irradiated colorectum of two murine strains.

PURPOSE: A strain difference in the development of radiation-induced fibrosis of the colorectum was recently observed. C57B1/6 mice developed colorectal obstruction with significantly higher incidence compared to C3Hf/Kam mice after partial volume irradiation with 30 Gy. Previous reports have demonstrated differences in cytokine mRNA levels in fibrosis-prone and -resistant mice after lung irradiation. The aims of this study are to determine if there are strain differences in: 1) the histology of the lesion, 2) mRNA levels for transforming growth factor beta (TGFbeta) isoforms and tumor necrosis factor alpha (TNFalpha), and 3) immunohistochemical staining patterns using antibodies against the TGFbeta isoforms and latency-associated peptide (LAP). METHODS AND MATERIALS: The colorectum of male C3Hf/Kam (C3H) and C57B1/6 (B6) mice were irradiated using a dose/length combination (30 Gy to 13.7 mm) that resulted in 10 or 100% incidence of obstruction by 6 months in each strain, respectively. Colorectal tissue was removed from 6 hours to 120 days after irradiation as well as from obstructed mice and prepared for histology, RNase protection assay, and immunofluorescence. RESULTS: Distinct differences in the histological phenotype for the two strains were observed at times preceding obstruction. Samples from B6 mice showed increased hyperplastic crypts, colitis cystica profunda, and fibrosis within the lamina propria, compared to identically treated C3H mice. Fibrosis in the lamina propria of B6 mice appeared early, beginning at 75 days after irradiation, and was progressive, whereas fibrosis in C3H mice appeared simultaneous with obstruction and may have been a reaction to ulceration. No consistent strain difference in mRNA levels for TGFbeta1, 2, 3 or TNFalpha were observed, although mRNA levels of TGFbeta1 and TNFalpha were significantly elevated in both strains relative to nonirradiated controls. Immunofluorescent staining for TGFbeta1, 3 and LAP was observed in hyperplastic crypts and colitis cystica profunda adjacent to regions of fibrosis, histological changes that were present predominately in the B6 strain. CONCLUSIONS: The response of the colorectum to irradiation involves changes in the expression of several different cytokines. However, the lack of a consistent strain difference in TGFbeta1, 2, 3 and TNFalpha mRNA levels, despite strain differences in both the incidence of colorectal obstruction and histological features preceding obstruction, suggests that mRNA changes in these fibrogenic cytokines are not the critical determinant of the strain difference and are not related to the process of radiation-induced colorectal fibrosis in these mouse strains. Strain-dependent differences were observed in the localization of active TGFbeta, but these differences were related to the histological changes specifically found in the irradiated colon of the B6 strain.

Murine strain differences in the volume effect and incidence of radiation-induced colorectal obstruction.

PURPOSE: Interindividual variation in the level of normal tissue damage after radiotherapy has been clinically observed. Murine models have suggested that there may be a genetic component to the variation in susceptibility of different radiation-induced normal tissue complications. Currently, there are no experimental data available describing interstrain differences in the "volume effect" for irradiated normal tissues, such as the colorectum. The aims of this study are to determine if there are strain differences in: 1. the incidence of colorectal obstruction; and 2. the volume effect, after irradiation of the colorectum using two mouse strains that are known to vary in their susceptibility for developing pulmonary fibrosis. METHODS AND MATERIALS: Various lengths (5.2 to 22.9 mm) of the colorectum of male C57B1/6 and C3Hf/Kam mice were irradiated with a single dose (30 Gy) of 137Cs gamma rays. Also, various doses (20 to 35 Gy) were given to a single length (22.9 mm) of colorectum. The incidence of obstruction was determined as a function of length and dose at 6 months after irradiation. The Threshold Probability model was fit to the length-response data. RESULTS: C57B1/6 mice developed colorectal obstruction at significantly higher incidence than C3Hf/Kam mice at all lengths after a single dose of 30 Gy. In addition, the data showed a strain difference in the threshold length of colorectum that had to be irradiated before obstructions were observed. CONCLUSION: Strain differences in the incidence of radiation-induced colorectal obstruction were observed, consistent with previous studies that showed a strain difference in radiation-induced pulmonary fibrosis. The presence of a threshold length of colorectum that was different for the two strains is consistent with the concept that there may be a critical threshold amount of colorectal tissue that can tolerate a high dose without complication, and that the dimensions of the threshold may vary among individuals.

Volume effects and epithelial regeneration in irradiated mouse colorectum.

The use of three-dimensional treatment planning and volume-reduction techniques in radiotherapy has prompted the development of a number of mathematical models to describe the effect of changing treatment volume on the probability of associated complications in normal tissues. However, limited data are available to test or support these models. One prediction of the Probability model and analogous models, which describe the volume-effect relationship for late end points in tissues with a series-type arrangement of functional subunits, is that there is no threshold volume in the development of the end point. This hypothesis was tested in mouse colorectum, a normal tissue with functional subunits suggested to be arranged in series, using the incidence of obstructions due to consequential fibrosis as the end point of damage. Various lengths of the colorectum of C3Hf/Kam mice were irradiated with single doses of 250 kVp X rays. A threshold length between 10 and 15 mm was observed after 32 Gy. The Probability model could not describe the data adequately, but a modified version that included a threshold volume term (the Threshold Probability model) provided an excellent fit. In a separate experiment, epithelial regeneration (migration, extracryptal proliferation and formation of new crypts) was examined as a possible mechanism for the threshold length. Re-epithelialization was complete after 32 Gy was delivered to lengths below (5 or 10 mm) but not above (20 mm) the threshold for consequential obstruction. Proliferation of epithelial cells outside the crypt on the mucosal surface (i.e. extracryptal proliferation) may contribute to the regeneration process. The data indicate that regeneration of the epithelium after irradiation results in a threshold length of the colorectum in the development of consequential fibrosis, in contradiction to predictions of the Probability model.

High dose rate (HDR) and low dose rate (LDR) interstitial irradiation (IRT) of the rat spinal cord.

PURPOSE: To describe a newly developed technique to study radiation tolerance of rat spinal cord to continuous interstitial irradiation (IRT) at different dose rates. MATERIAL AND METHODS: Two parallel catheters are inserted just laterally on each side of the vertebral bodies from the level of Th10 to L4. These catheters are afterloaded with two 192Ir wires of 4 cm length each (activity 1-2.3 mCi/cm) for the low dose rate (LDR) IRT or connected to the HDR micro-Selectron for the high dose rate (HDR) IRT. Spinal cord target volume is located at the level of Th12-L2. Due to the rapid dose fall-off around the implanted sources, a dose inhomogeneity across the spinal cord thickness is obtained in the dorso-ventral direction. Using the 100% reference dose (rate) at the ventral side of the spinal cord to prescribe the dose, experiments have been carried out to obtain complete dose response curves at average dose rates of 0.49, 0.96 and 120 Gy/h. Paralysis of the hind-legs after 5-6 months and histopathological examination of the spinal cord of each irradiated rat are used as experimental endpoints. RESULTS: The histopathological damage seen after irradiation is clearly reflected the inhomogeneous dose distribution around the implanted catheters, with the damage predominantly located in the dorsal tract of the cord or dorsal roots. With each reduction in average dose rate, spinal cord radiation tolerance is significantly increased. When the dose is prescribed at the 100% reference dose rate, the ED50 (induction of paresis in 50% of the animals) for the HDR-IRT is 17.3 Gy. If the average dose rate is reduced from 120 Gy/h to 0.96 or 0.49 Gy/h, a 2.9- or 4.7-fold increase in the ED50 values to 50.3 Gy and 80.9 Gy is observed; for the dose prescribed at the 150% reference dose rate (dorsal side of cord) ED50 values are 26.0, 75.5 and 121.4 Gy, respectively. Using different types of analysis and in dependence of the dose prescription and reference dose rate, the alpha/beta ratio varies between 1.46 (0.06-3.08 CL) and 2.17 Gy (0.08-4.61). The half time of repair during continuous irradiation is 1.76 h (1.33-2.64), while no indication is found for a biphasic pattern of the kinetics of repair. CONCLUSION: The implantation technique in our study has shown to be a reliable model to compare the effectiveness of HDR- and LDR-interstitial continuous irradiation at different dose rates.

Substructure in the radiation survival response at low dose in cells of human tumor cell lines.

In earlier studies using asynchronously growing Chinese hamster cells, we observed substructure in the survival response at low doses. The substructure appeared to result from subpopulations of cells having different, cell cycle phase-dependent radiosensitivity. We have now applied the same flow cytometry and cell sorting technique to accurately measure the responses of cells of eight different asynchronously growing human tumor cell lines, representing a wide range in radiosensitivity. When the data were fitted with a linear-quadratic (LQ) function, most of these lines showed substructure similar to that observed in Chinese hamster cells, with the result that values of alpha and beta were dependent on the dose range used for fitting. Values of alpha describing the low-dose response were typically smaller (by as much as 2.2 times) than the alpha describing the high-dose response, while values of beta were larger at low doses. Values of alpha/beta from our measurements are in reasonable agreement with other values published recently if we fit the data for the high-dose range (excluding, for example, 0-4 Gy), which corresponds to a conventional survival response measurement. However, the values of alpha/beta describing the low-dose range were, on average, 2.8-fold smaller. The results show that the usual laboratory measurement of cell survival over 2 or 3 logs of cell killing, if fitted with a single LQ function, will yield alpha and beta values which may give a rather poor description of cell inactivation at low dose in asynchronous cells, no matter how carefully those measurements are done, unless the low-dose range is fitted separately. The contribution of killing represented by the beta coefficient at low doses was found to be surprisingly large, accounting for 40-70% of cell inactivation at 2 Gy in these cell lines. A two-population LQ model provides excellent fits to the data for most of the cell lines though, as one might expect with a five-parameter model, the best-fitting value of the various parameters is far from unique, and the values are probably not reliable indicators of the size and radiosensitivity of the different cell subpopulations. At very low dose, below 0.5-1 Gy, another order of substructure is observed: the hypersensitive response; this is described in the accompanying paper (Wouters et al., Radiat. Res. 146, 399-413, 1996).

The use of radiochromic film to measure dose distributions resulting from high dose rate 192Iridium single catheter treatments.

PURPOSE: Radiochromic film was used to measure and compare the dose distributions parallel to a high dose rate (HDR) 192Iridium (192Ir) brachytherapy afterloading catheter that resulted from optimized treatment plans using various combinations of prescribed dose magnitude and location as well as source spacing. METHODS AND MATERIALS: Differences exist among clinical investigators for specification of the magnitude and location of prescribed treatment dose for brachytherapy irradiations using HDR 192Ir afterloading. Typical prescriptions for endobronchial irradiation include 5 to 10 Gy at 10 mm or 15 Gy at 6 mm measured from the center of the afterloading catheter. The dose distributions that result from these irradiations are very difficult to quantify by conventional dosimetry methods. This study used radiochromic film to measure the dose distributions resulting from optimized treatment plans for source dwell position separations of 2.5 or 5.0 mm and for a prescribed treatment dose of either 15 Gy at 6 mm or 5 Gy at 10 mm, conditions that have been used at M. D. Anderson Cancer Center for the treatment of endobronchial lesions. An acrylic phantom was designed to allow for measurement of the dose distributions at 0.95 mm (catheter surface), 6 mm, and 10 mm from and parallel to the catheter for sources positioned along either 20 or 80 mm of the catheter. RESULTS: Radiochromic film is shown to be a suitable quality assurance and dosimetry modality for the measurement of the dose distribution along an afterloading catheter resulting from an HDR I92Ir source. Each of the treatment plans was about equally effective in being able to produce a uniform dose distribution at their respective planned target distances. Differences were more apparent when comparing the dose distributions at nontargeted distances. On the catheter surface the dose was very nonuniform and in the case of 2.5 mm source spacing along 20 mm of catheter with target dose planned to 10 mm, the central minimum dose was only 13 to 24% of the dose opposite to the most proximal and distal sources. The absolute doses measured at equivalent distances for the 15 Gy planned to 6 mm treatments are about 1.3 to 1.5 times higher than those measured for the 5 Gy planned to 10 mm treatments. It was also observed that the lateral positioning of the encapsulated source within the afterloading catheter can contribute to dose differences about the catheter that are greatest for measurements made in contact with the catheter surface (24 to 40%) but may also be large at the treatment planning distances of 6 (0 to 15%) and 10 mm (0 to 9%). CONCLUSION: At their respective treatment planning distances of 6 or 10 mm, each of the treatment plans produced dose distributions of comparable uniformity. Against the catheter, relatively more uniform dose distributions with higher minimum doses were obtained for (a) dose prescription at 6 mm, rather than at 10 mm; (b) source separation of 2.5 mm, rather than 5.0 mm (except for a 20 mm active catheter length with dose planned to 10 mm); and (c) longer active length of the catheter of 80 mm, rather than 20 mm.

The cytotoxicity of melphalan and its relationship to pH, hypoxia and drug uptake.

In the present in vitro studies we examined the effect of hypoxia and acidic pH, two important consequences of reduced blood flow in vivo, on the cytotoxicity of melphalan treatment in Chinese hamster V79-WNRE and SiHa human tumor cells. Cells were exposed to various concentrations of melphalan for 1 hr at 37 degrees C under oxic or hypoxic conditions; pH 6.6 or 7.4, and cell survival was measured. The cytotoxicity of melphalan was potentiated by both low pH and hypoxia, in both cell lines. The overall potentiation, expressed as an enhancement ratio (ER), from both hypoxia and low pH was 3.5 in V79-WNRE and 2.9 in SiHa cells. The potentiation of cell killing produced by hypoxia alone (ERHyp) ranged from 1.4 to 1.9, and was greater in V79-WNRE than in SiHa cells. The potentiation from low pH (ERpH) was approximately 2 in both cell lines. HPLC analysis showed substantial intracellular accumulation of melphalan in both cell lines. Hypoxia and reduced pH further enhanced uptake of melphalan but this was not sufficient by itself to account for the increased potentiation of cytotoxicity observed under those conditions.

The effect of low pH and hypoxia on the cytotoxic effects of SR4233 and mitomycin C in vitro.

PURPOSE: We examined the effect of acidic pH and hypoxia on the cytotoxicity of SR4233 and mitomycin C in vitro. METHODS AND MATERIALS: The importance of tumor microenvironment to the response of solid tumors to cytotoxic treatment is well established. The bioreductive drug SR4233 has a very substantial selective toxicity for hypoxic cells. We have used both Chinese hamster and human tumor cells to investigate the influence of low pH and hypoxia on the response of cultured cells to treatment with SR4233 or mitomycin C. RESULTS: We found that low pH (6.6) had little effect on the hypoxic toxicity of SR4233; under aerobic conditions, however, low pH substantially increased the cytotoxic effects of 1 h exposure to SR4233, with drug dose enhancement ratios (ER) of 3.9 and 2.5 in V79 and HT-29 cells, respectively. In similar studies with mitomycin C, hypoxia had little effect on the cytotoxicity of mitomycin C in V79 cells, though a low pH of 6.6 enhanced the cytotoxicity under both aerobic and hypoxic conditions (ER approximately 2). In HT-29 cells, neither low pH nor hypoxia had any significant effect on mitomycin C toxicity. CONCLUSION: Low pH, like hypoxia, is a common feature of solid tumors and can be an important determinant of the cytotoxic effect of bioreductive drugs such as SR4233 and mitomycin C.

The survival of asynchronous V79 cells at low radiation doses: modeling the response of mixed cell populations.

We have observed that when a single linear-quadratic (LQ) function is used to fit the radiation survival response of an asynchronously dividing population of V79 cells, a consistent misfit occurs at low doses. The data can be better described by fitting the low-dose and high-dose ranges separately, and there is evidence of a two-component response. The most obvious explanation is that we may simply be seeing the response of subpopulations of cells of different radiosensitivity: sensitive G1-, G2- and M-phase cells and resistant S-phase cells. The cell sorting assay for cell survival which we have used in these studies may thus be providing sufficient accuracy to resolve these subpopulations, not previously seen in conventional survival measurements. An alternative explanation is that the linear-quadratic function may be inappropriate for accurate description of the radiation survival response at low dose, at least for these cells. To test this hypothesis we have used three other models to fit the data: the single-hit plus multi-target (SHMT) model and the two-parameter repair-misrepair (RMR) model both yielded inferior fits to the asynchronous survival data; the three-parameter RMR model provided an improved fit to the data. The best fit, however, was obtained using a two-population LQ model, which suggested approximately equal numbers of sensitive and resistant cells. When the survival response of tightly synchronized G1/S-phase cells was measured using the cell sorting assay, no substructure was observed. This offers strong support to the hypothesis that the substructure observed in the asynchronous survival response is due to subpopulations of cells of different, cycle-dependent radiosensitivity.

Substructure in the radiation survival response at low dose: asynchronous and partially synchronized V79-WNRE cells.

The radiation survival response of asynchronously-dividing populations of the cell line V79-WNRE indicates substructure in the low dose region that can be better characterized by separately fitting the data within the low and high dose regions to the linear-quadratic (LQ) equation. Flow cytometry and cell sorting techniques have been used to determine the response of both asynchronous populations and synchronous populations obtained by mitotic selection with or without an additional drug block. The statistically significant substructure which is present in the radiation response of asynchronous cells is absent in G1/S cells synchronized by mitotic selection followed by hydroxyurea (or aphidicolin) accumulation at the G1/S boundary. Such G1/S populations have a radiation response that is well characterized by a single LQ expression over 3 logs of cell inactivation. In contrast, the radiation response of cells synchronized by mitotic selection alone and irradiated in early G1 shows substantial substructure when fitted to the LQ equation, reflecting some radioresistance at high dose. While this response is well described by a single hit plus multitarget equation, it is possible that the small proportion (< 5%) of interphase cells in such mitotically-selected populations may be responsible for the resistance observed at higher doses.

The effect of pH on the aerobic and hypoxic cytotoxicity of SR4233 in HT-29 cells.

We have observed that low pH can substantially potentiate the cytotoxic effect of the bioreductive drug SR4233 in aerobic HT-29 human tumour cells. No such potentiation was observed under hypoxic conditions. This pH effect might be relevant both to the therapeutic effectiveness and to the normal tissue toxicity of this new agent.

The effect of hypoxia and low pH on the cytotoxicity of chlorambucil.

Studies with mouse tumors have shown that the effectiveness of certain chemotherapeutic agents can be enhanced if they are used in appropriate combination with an anti-hypertensive drug such as hydralazine. This results in reduced tumor blood flow with, among other things, a consequent decrease in oxygenation and increase in acidity in the tumor tissue. The purpose of the present work was to determine to what extent hypoxia and low pH are involved in the mechanism of this effect for chlorambucil. V79-WNRE cells were exposed to various drug concentrations under aerobic or hypoxic conditions, pH 6.4 or 7.4. Measurements of cell survival following 1 hr exposure at 37 degrees C showed that pH 6.4 produced a large potentiation of cell killing by chlorambucil (ER = 4 approx.); hypoxia, on the other hand, had little effect. The potentiation was shown to be greatest for pH values below 7.0. HPLC measurements of drug uptake were made since it was anticipated that chlorambucil, a weak acid, might tend to accumulate in cells under conditions of low extracellular pH. It was found that at an extracellular pH of 6.4 the ratio of the intracellular (Ci) and extracellular (Ce) drug concentrations was increased 4.5 and 3.6 fold for aerobic and hypoxic conditions, respectively. This probably explains most, if not all, of the cell killing potentiation observed at low pH.