Condensed chromatin and cell inactivation by single-hit kinetics.

Mammalian cells are extremely sensitive to gamma rays at mitosis, the time at which their chromatin is maximally condensed. The radiation-induced killing of mitotic cells is well described by single-hit inactivation kinetics. To investigate if radiation hypersensitivity by single-hit inactivation correlated with chromatin condensation, Chinese hamster ovary (CHO) K1 (wild-type) and xrs-5 (radiosensitive mutant) cells were synchronized by mitotic shake-off procedures and the densities of their chromatin cross sections and their radiosensitivities were measured immediately and 2 h into G1 phase. The chromatin of G1-phase CHO K1 cells was dispersed uniformly throughout their nuclei, and its average density was at least three times less than in the chromosomes of mitotic CHO K1 cells. The alpha-inactivation co-efficient of mitotic CHO K1 cells was approximately 2.0 Gy(-1) and decreased approximately 10-fold when cells entered G1 phase. The density of chromatin in CHO xrs-5 cell chromosomes at mitosis was greater than in CHO K1 cell chromosomes, and the radiosensitivity of mitotic CHO xrs-5 cells was the greatest with alpha = 5.1 Gy(-1). In G1 phase, CHO xrs-5 cells were slightly more resistant to radiation than when in mitosis, but a significant proportion of their chromatin was found to remain in condensed form adjacent to the nuclear membrane. These studies indicate that in addition to their known defects in DNA repair and V(D)J recombination, CHO xrs-5 cells may also be defective in some process associated with the condensation and/or dispersion of chromatin at mitosis. Their radiation hypersensitivity could result, in part, from their DNA remaining in compacted form during interphase. The condensation status of DNA in other mammalian cells could define their intrinsic radiosensitivity by single-hit inactivation, the mechanism of cell killing which dominates at the dose fraction size (1.8-2.0 Gy) most commonly used in radiotherapy.

Radiation fields backscattered from material interfaces: I. Biological effectiveness.

Confluent cultures of CHO-K1 and CHO-xrs5 cells were irradiated attached to 6 microm Mylar with 137Cs gamma rays and 200 kVp X rays adjacent to scattering materials consisting of polystyrene, glass, aluminum, copper, tin and lead. The absorbed dose in cell nuclei was estimated from measurements of backscattered dose made with a parallel-plate ion chamber with a 5-microm Mylar window and a gas volume whose thickness was equivalent to approximately 2.6 microm of cells or tissue. Cell inactivation after various doses was measured by clonogenic assays after trypsinization and enumeration. Survival curves constructed from data pooled from at least two independent experiments were best fitted to a linear-quadratic (LQ) or a linear equation for CHO-K1 and CHO-xrs5 cells, respectively. An average distance of 9.3+/-1.9 microm from the scattering surfaces to the midline of nuclei for both the cell lines was estimated from electron micrographs of fixed cell sections. The major differences in biological effect observed when the cells were irradiated adjacent to these materials could be largely explained by the differences in the physical dose. Further analyses using the LQ equation suggested additional biological effects with implications for the mechanisms involved. CHO-K1 cells showed a small but consistent increase in the low-dose (alpha-inactivation coefficient) mechanism for both radiations scattered from high-Z material. An increased value of the alpha coefficient suggests an increase in RBE which could be associated with a higher proportion of low-energy and track-end electrons in these fields. The radiation fields which produced maximum single-hit killing in CHO-K1 cells also produced less killing by the quadratic (beta-inactivation coefficient) mechanism. In contrast, when similarly irradiated, CHO-xrs5 cells exhibited significantly lower alpha coefficients of inactivation. The mechanistic basis for this opposite effect of backscattered radiations in these cell lines is as yet unknown.

Microdosimetric single event spectra of Ytterbium-169 compared with commonly used brachytherapy sources and teletherapy beams.

Ytterbium-169 has been developed as a possible replacement for Iridium-192 and Iodine-125. The Theory of Dual Radiation Action predicts that the initial slope of the cell survival curve and therefore the relative biological effect at low dose rate is proportional to dose average lineal energy, yd, which is the microscopic analog of the dose average linear energy transferred. The quality factor used in radiation protection has been shown to be a function of the frequency average lineal energy, yf. Single event microdosimetric spectra for 60Co, 137Cs, 192Ir, 125I and 169Yb were measured in air and at several depths in phantom with a Rossi proportional counter. These spectra show marked differences between sources. The microscopic analogs of the track average and dose average LET, (yd and yf, respectively) differ between isotopes by factors of two or even higher in comparison to megavoltage electron beams. These yd's and yf's for 169Yb are consistently higher when compared to 60Co or 137Cs but are approximately equal to those for 125I. Values of yf and yd for 192Ir are intermediate between 60Co and 169Yb. The Theory of Dual Radiation Action predicts a low dose rate RBE (assuming a 1 micron effective site diameter) compared to 60Co (in air) of: 1.00 for 137Cs, 1.29 for 192Ir, 1.60 for 169Yb and 1.77 for 125I.

Calculated risk of breast cancer following mantle irradiation determined by measured dose.

Tumor registry data indicate a two- to fourfold increased incidence of breast cancer following mantle irradiation, but cumulative risk is unknown. Radiation exposure to the breasts underlying the mantle block ranges from 4 to 40 Gy and is dependent on relative positions of the breasts and mantle block. Unshielded outer breast quadrants near axillary nodal regions receive 36 to 40 Gy, while central breast quadrants under the lung blocks receive approximately 4 Gy as determined by dose volume histogram analysis. Relative dose risk analysis for breast cancer following mantle irradiation was performed and indicated an overall excess risk of 1.5 for the upper outer quadrant (total dose 40 Gy), 1.3 for the upper and lower inner, and central quadrants (total dose 15 to 20 Gy), and 1.2 for the lower outer quadrant (total dose 4 Gy). Linear and cell-kill carcinogenesis models demonstrated similar relative risk assessments in the low-dose regions, defined as < 15 Gy. Predicted risk for breast cancer in the high-dose regions (> or = 15 Gy) varied considerably according to the model evaluated. The linear model predicted a three to ten times greater risk above baseline breast cancer incidence for the high-dose regions. In contrast, the cell-kill model predicted no excess cases of breast cancer, assuming cell death at these higher dose levels. The greatest relative predicted risk is observed in women < 20 years of age at the time of irradiation; however, women older than 20 years continue to have a 50% higher than baseline risk for subsequent breast cancer development. All women treated for Hodgkin's lymphoma should undergo dose volume histogram evaluation. Prospective clinical and mammographic evaluations should be performed in all female patients following mantle irradiation to better define the risk for secondary breast carcinogenesis.

Is the dose-response relationship for local control of Hodgkin's disease obscured by lung inhomogeneity corrections?

Absence of a dose-response relationship has been reported for local control of Hodgkin's disease between 30 and 42 Gy delivered to the mantle field in definitive irradiation. These dose ranges were determined at the central ray using irregular field (IF) calculations without benefit of dosimetric correction for lung inhomogeneity. Detailed analysis was performed of dose delivered to hilar and mediastinal regions with mantle field irradiation incorporating lung inhomogeneity corrections for the indicated dose ranges using 60Co and 6 MV linear accelerator. Inclusion of lung inhomogeneity (LI) corrections revealed dose heterogeneity within the treatment volume, delivering higher total doses to hilar and lateral mediastinal regions. For a prescribed dose of 40 Gy to midline with 6 MV photons, the hila and mediastinum would receive 45 Gy with IF and LI corrections. Similar increases are observed with 60Co. Mantle field calculations should be performed with CT planning including lung inhomogeneity corrections to account for anatomic irregularity of the mediastinal contour and interposed lung. As lung inhomogeneity dosimetric data become available, conclusions drawn from clinical experience should be re-evaluated based upon location and extent of mediastinal disease involvement. Implications for treatment associated late toxicity using dose-volume histogram analysis should be considered.

Dosimetry of the breast for determining carcinogenic risk in mantle irradiation.

Development of secondary malignancies following treatment of Hodgkin's disease with radiation (central axis midline dose of 3600-4500 cGy) is a recognized risk, and the incidence in breast cancer has been reported to increase by a factor of 4.3 (95% confidence level 2.0 to 8.4) for patients treated with mantle irradiation. Increased incidence of breast cancer has also been shown in atomic bomb survivors, women who underwent multiple fluoroscopic examinations, and women treated for postpartum mastitis. The dose response, however, for radiation dose above 1000 cGy is virtually unknown. Quantitative analysis of carcinogenesis after radiation is exacerbated by the large dose gradient across the breast (300-4200 cGy for midline doses of 4000 cGy), large individual variation in breast size and treatment field position. We have developed differential dose volume histograms calculated using a 3-dimensional (Eg TAR) algorithm as a potential tool for retrospective and prospective epidemiological evaluation. The breast volume forms a bimodal distribution with respect to dose and with further analysis other quantities, such as mean dose and integral dose, can be calculated from the histograms. Using the mean dose, the linear model for carcinogenesis predicts an increased incidence of secondary breast cancer by a factor of 11.6 and 9.6 for the left and right breast, respectively. The dose calculations has been corrected for inhomonogenities 3-dimensionally and test of the accuracy has been included.

Comparison of dose distribution by means of bolus versus water bath phantom techniques in treatment of cutaneous sarcoma of an extremity.

Cutaneous involvement by Kaposi's sarcoma (KS) is frequently diffuse, requiring large field irradiation. Selective tissue compensation techniques, accomplished with either a 1.5 cm bolus cast or water bath phantom, are compared. Dosimetric determinations were performed using 6 MVX with bone heterogeneity corrections. Application of 1.5 cm bolus effectively eliminated dose buildup and provided tissue compensation equivalent to water bath phantom technique. Treatment of other cutaneous malignancies, such as angiosarcoma, requiring higher total doses, should be accompanied by dosimetric evaluation, because cutaneous and midline dose inhomogeneity may result in a significant total dose differential over the target volume. Application of 1.5 cm bolus improved accuracy in treatment positioning, eliminated concerns regarding infection control in AIDS-related KS, and provided dose homogeneity comparable to the more standard water bath phantom technique.

Microdosimetric single-event spectra for megavoltage electrons.

Experimental techniques have been developed for obtaining single-event microdosimetric spectra from hospital based linear accelerators. Therapeutic electrons of 12, 15, 18, and 20 MeV from Clinic 18 and 20 accelerators have been produced at ultralow dose rates. Details of the experimental methods have been described previously by the authors. Single-event lineal energy spectra for these beams, as measured by a Rossi chamber differ significantly from cobalt-60 both in shape and dose average lineal energy (yd) which, depending on electron energy, can be 20-40% lower. The spectral peaks for electrons are greatly enhanced compared to cobalt. The yd for electrons also differs from previously reported values for 10-15 MV photon beams. These differences are less pronounced than for differences with cobalt. Spectral peaks and shapes correlate well with the actual electron stopping powers in tissue. The theory of dual radiation action predicts changes in relative biological effectiveness at low doses for megavoltage photon and electrons. Although at clinical doses the predicted differences are not statistically significant.

Microdosimetry of 10-15 MeV bremsstrahlung x rays.

Experimental techniques have been developed for obtaining microdosimetric spectra on a hospital-based linear accelerator. Teletherapy beams of 10 and 15 MeV bremsstrahlung x rays from a Varian Clinac-18 and Clinac-20, respectively, have been produced at ultralow dose rates (50-200 microGy/h) which enables direct measurements of lineal energy distributions with a conventional Rossi-type gas proportional counter. Extensive measurements have been made to insure that the dosimetric properties of these low dose rate beams are nearly identical to those produced under high dose rate clinical conditions. Analytical procedures have been developed to correct measured lineal energy spectra for pileup caused by the low duty factor of the linear accelerator. The lineal energy spectra of these megavoltage beams differ significantly from Co-60, with dose averaged lineal energies (yD) being 20%-30% lower than for Co-60. Although such differences may not be important at clinical doses, the theory of dual radiation action does predict a lower biological effectiveness for these beams at very low dose levels.

Surface dose for megavoltage photon beams outside the treatment field.

Measurements made on photon beams from four different radiotherapy machines have demonstrated that skin dose several centimeters outside the boundary of a treatment field may be as much as 20% of the central axis maximum dose. This surface dose has been measured for an AECL Theratron 80, Siemens Mevatron VI, Varian Clinac 20, and CGR Sagittaire for distances up to 12 cm outside the field boundary and for depths up to the depth of maximum central axis dose. This dose has also been measured as a function of field size and of source-to-skin distance. For the lower energy photon beams, this radiation is significantly attenuated in the first 2-3 mm of tissue, while for higher energy beams, a buildup phenomenon with a dmax of 2-3 mm is observed. The magnitude of this radiation is approximately linearly dependent upon field dimension for all energies.