Selective elimination of a chemoresistant side population of B-CLL cells by cytotoxic T lymphocytes in subjects receiving an autologous hCD40L/IL-2 tumor vaccine.

Side-population (SP) analysis identifies precursor cells in normal and malignant tissues. Cells with this phenotype have increased resistance to many cytotoxic agents, and may represent a primary drug-resistant population in malignant diseases. To discover whether drug-resistant malignant SP cells are nonetheless sensitive to immune-mediated killing, we first established the presence of a malignant CD5(+)CD19(+) SP subset in the blood of 18/21 subjects with B-cell chronic lymphocytic leukemia (B-CLL). We examined the fate of these cells in six of these individuals who received autologous human CD40 ligand and interleukin-2 (hCD40L/IL-2) gene-modified tumor cells as part of a tumor vaccine study. Vaccinated patients showed an increase in B-CLL-reactive T cells followed by a corresponding decline in circulating CD5(+)CD19(+) SP cells. T-cell lines and clones generated from vaccinated patients specifically recognized B-CLL SP tumor cells. Elimination of SP cells is likely triggered by their increased expression of target antigens, such as receptor for hyaluronan-mediated motility (RHAMM), after stimulation of the malignant cells by hCD40L, as CD8(+) RHAMM-specific T cells could be detected in the peripheral blood of immunized patients and were associated with the decline in B-CLL SP cells. Hence, malignant B cells with a primary drug-resistant phenotype can be targeted by T- cell-mediated effector activity after immunization of human subjects.

A distinct "side population" of cells in human tumor cells: implications for tumor biology and therapy.

Stem cells have an extensive capacity to proliferate, differentiate and self-renew. In many mammals, including humans, an adult stem cell subpopulation termed the "side population" (SP) has been identified. SP cells can rapidly efflux lipophilic fluorescent dyes to produce a characteristic profile based on fluorescence-activated flow cytometric analysis. Previous studies have demonstrated SP cells in bone marrow obtained from patients with acute myeloid leukemia, suggesting that these cells might be candidate leukemic stem cells, and recent studies have found a SP of tumor progenitor cells in human solid tumors. These new data indicate that the ability of malignant SP cells to expel anticancer drugs may directly improve their survival and sustain their clonogenicity during exposure to cytostatic drugs, allowing disease recurrence when therapy is withdrawn. Identification of a tumor progenitor population with intrinsic mechanisms for cytostatic drug resistance might also provide clues for improved therapeutic intervention.

A distinct "side population" of cells with high drug efflux capacity in human tumor cells.

A subset of stem cells, termed the "side population" (SP), has been identified in several tissues in mammalian species. These cells maintain a high efflux capability for antimitotic drugs. We have investigated whether functionally equivalent stem cells also may be detected in human cancers. We initially examined primary tumor cells from 23 patients with neuroblastoma and cell lines derived from a range of other tumors. A distinct SP was found in neuroblastoma cells from 15 of 23 patients (65%). The SP was capable of sustained expansion ex vivo and showed evidence for asymmetric division, generating both SP and non-SP progeny. These cells also expressed high levels of ABCG2 and ABCA3 transporter genes and had a greater capacity to expel cytotoxic drugs, such as mitoxantrone, resulting in better survival. A SP also was detected in breast cancer, lung cancer, and glioblastoma cell lines, suggesting that this phenotype defines a class of cancer stem cells with inherently high resistance to chemotherapeutic agents that should be targeted during the treatment of malignant disease.

Comparison of commercially available three-dimensional treatment planning algorithms for monitor unit calculations in the presence of heterogeneities.

This study uses an anthropomorphic phantom and its computed tomography (CT) data set to evaluate monitor unit (MU) calculations using the CMS Focus Clarkson, the CMS Focus Multigrid Superposition Model, the CMS Focus FFT Convolution Model, and the ADAC Pinnacle Collapsed Cone Convolution Superposition Algorithms. Using heterogeneity corrections, a treatment plan and corresponding MU calculations were generated for several typical clinical situations. A diode detector, placed in an anthropomorphic phantom, was used to compare the treatment planning algorithms' predicted doses with measured data. Differences between diode measurements and the algorithms' calculations were within reasonable levels of acceptability as recommended by Van Dyk et al. [Int. J. Rad. Onc. Biol. Phys. 26, 261-273 (1993)], except for the CMS Clarkson algorithm, which predicted too few MU for delivery of the intended dose to chest wall fields.

Modification of a linear accelerator table top for non-coplanar conformal brain radiotherapy.

The use of non-coplanar conformal therapy necessitates the use of unusual beam projections that may not be accomplished with a conventional linear accelerator table top. Modification of the table top can increase the available combinations of gantry and couch rotation. A standard Philips table top, supplied with an SL 75-5 linear accelerator, was modified to increase available combinations of gantry and couch rotation. This was accomplished by shortening the length and decreasing the width of the table top. The modified table top increases the combinations of gantry and couch angles significantly, simplifying the delivery of non-coplanar conformal therapy without significant compromise to routine treatment. The modification of a standard linear accelerator table top has increased the available combinations of gantry and couch rotation to accommodate non-coplanar conforrmal radiotherapy.

Comparison of effectiveness of thermoluminescent crystals LiF:Mg,Ti, and LiF:Mg,Cu,P for clinical dosimetry.

This study compared the relative effectiveness of TLD crystals LiF:Mg,Ti (TLD-100) and LiF:Mg,Cu,P (TLD-700H) for clinical dosimetry, focusing on reproducibility, linearity, and energy response. Experimental results indicated that TLD-700H was superior to TLD-100 with regard to reproducibility, lack of supralinearity, and the absence of variation in TL signal with radiation quality. TLD-700H also had the additional advantages of higher sensitivity and immediate readability. The investigators conclude that this relatively new TLD crystal shows promising potential for clinical dosimetry.

A self-collimating convolution backprojection algorithm for optimizing dose distributions of I-125 prostate implants.

The algorithm presented here for optimizing brachytherapy dose distributions is based on the idea that the seed distribution can be modeled as an activity distribution determined analogously to gamma camera imaging. The peripheral dose to the tumor is converted to a set of uncollimated projection data that are then filtered and backprojected to produce an initial seed distribution. The actual doses resulting from the seed placement are used to correct the initial projection data for attenuation, scatter, and lack of collimation. The corrected projection data are backprojected a second time to yield the optimized but unconstrained seed distribution. Clinical constraints such as the number of different seed activities, the maximum seed activity, the minimum peripheral tumor dose, and the minimum percentage of the volume which receives less than a specified dose are then applied to the unconstrained solution. Through the entire process, the dose calculations are functions of source anisotropy, scatter, and attenuation. When applied to a set of elliptical contours, the algorithm produces elliptical peripheral dose isodose contours and reasonable dose volume histograms for a constrained solution. The results for actual patient prostate contours were not as good, primarily because of the difficulties encountered in dealing with the irregular geometry of the prostate. However, the algorithm shows promise for further research.

Effect of in vivo exposure to iodine-131 on the frequency and persistence of micronuclei in human lymphocytes.

The validity of the micronucleus test as a biomarker of chromosome damage in dividing mammalian cells is well established. This assay was used to study the response of peripheral lymphocytes of a 34-yr-old male patient following treatment with 131I ablative radiation therapy following a total thyroidectomy. Coincidentally, 8 mo before diagnosis, the patient had provided a blood sample for an in vitro study of micronucleus induction following exposure to graded doses of x-rays. The background frequency in the unexposed culture showed a mean count of 6.0 micronuclei per 1000 binucleated (first division) lymphocytes, while mean values of 18.5, 29.0, 41.0, 61.0 and 75.5 micronuclei/1000 cells were observed following x-ray doses of 5, 10, 15, 20, and 25 cGy, respectively. These data fit a nonthreshold, linear dose-response function (y = 2.78x + 3.71; r = .99). Eight months after the in vitro x-ray study, the subject was diagnosed with thyroid cancer. Surgery was performed, and 5 wk later the patient received 1.78 GBq (48 mCi) of 131I as adjuvant radiation therapy. Blood was drawn 11 d after the radiation treatment and at monthly intervals thereafter to analyze the frequency and persistence of micronuclei. The first posttreatment sample showed 35.5 micronuclei per 1000 binucleate cells. Based on the linear dose-response equation from the earlier study, the sixfold increase in micronucleus frequency suggests a dose to the peripheral blood of approximately 11 cGy. The cytogenetic dose estimate compares to approximately 30 cGy using a new model based on external whole-body counting data. Nine consecutive monthly samples have been analyzed to date. Although the micronucleus count has fluctuated (four- to sixfold above background), the frequency after 8 mo is equivalent to the first posttreatment sample. Data show that radiation-induced cellular lesions persist for months following relatively brief radiation exposure to a medical isotope. Results of this study support the conclusion that the lymphocyte micronucleus test is a rapid, sensitive, and perhaps quantitative biomarker of low-dose (< 25 cGy) radiation exposure.

The modification of specific absorption rates in interstitial microwave hyperthermia via tissue-equivalent material bolus.

Patterns of specific absorption rates generated by interstitial, microwave antenna arrays must be experimentally ascertained and quantified to facilitate their clinical incorporation. Phantom studies involved the use of four single-gap, coaxial antennas oriented in a 2 cm square array. These dipoles were driven in phase by a microwave generator at a frequency of 915 MHz. The inherent limitations in modifying the specific absorption rate patterns were addressed with the addition of bolus to the phantom. These additions of Guy's muscle tissue-equivalent material were made either proximal or distal to the phantom proper. Experiments conducted in the presence and absence of tissue-equivalent material bolus showed the ability to achieve broader bands of 50% power deposition in certain bolus conditions. These heating patterns were sufficiently reproducible and predictable to warrant clinical application of the bolus addition. A through-and-through method of catheter implantation allowed for bolus addition when deemed necessary. Treatments with veterinary and human patients using the bolus method to modify heating patterns yielded augmented patterns of power deposition. The effective length of the antennas that would radiate efficiently was essentially broadened via introduction of a microwave-interacting medium. As a result of the tissue equivalent material's ability to absorb microwave power, it was necessary to interpose minimally-interactive styrofoam spacers to limit heat transfer effects at the tissue-bolus interfaces.

A technique for delivery of total body irradiation for bone marrow transplantation in adults and adolescents.

With the increasing use of bone marrow transplantation for cancer, total body irradiation is becoming a more commonplace procedure in many of the larger centers across the country. The technical difficulties in delivering homogenous doses of radiation to the whole body are significant and involve many factors such as creation of a homogeneous, "flat" beam of radiation, and dealing with variations in patient thickness and tissue homogeneity, particularly in the lung. In addition, techniques must be used to safely and efficiently deal with patients who are usually very ill and require long treatment times. Although there is often an advantage in terms of dosimetry to using an AP/PA treatment technique, many institutions use parallel opposed lateral beams because of equipment and facility limitations. A technique has been devised that enables total body irradation to be given by an AP/PA technique using equipment available in many radiotherapy departments. Patients are supported in an upright position during treatment by means of a modified harness attached to the ceiling of the treatment room. Lung compensators are fixed to individually fitted "vests," allowing the patient moderate amounts of movement during treatment while maintaining the position of the compensator relative to the lungs. Thermoluminiscent dosimeter (TLD) dose measurements in a phantom indicate that this system can deliver accurate and homogeneous doses to lung tissue, while allowing a good degree of patient comfort and safety during the long treatment times that are required.

Incorporation of patient immobilization, tissue compensation and matchline junction technique for three-field breast treatment.

A protocol for the treatment of the intact breast was developed to maximize dose homogeneity and reproducibility. This protocol uses patient and breast immobilization, three-dimensional tissue compensators, and a technique for geometric matching of fields when the supraclavicular area is treated. A series of phantom measurements and analysis of patient port films was performed to evaluate dose homogeneity and reproducibility using this technique, and the potential adverse effect of loss of skin sparing from the immobilization device was investigated. Dose homogeneity throughout the phantom breast was within +/- 6% of the prescribed central axis dose, and homogeneity at the supraclavicular match line was +/- 10%. This represented a significant improvement over techniques not using tissue compensation or geometrically matched fields. Reproducibility of patient treatments was not significantly improved from previous non-immobilized treatment techniques, but there was no loss of skin sparing from the device, and other advantages of immobilization were observed. Details of the protocol are discussed together with changes that are currently being made to improve the results obtained thus far.

The distribution of power and heat produced by interstitial microwave antenna arrays: II. The role of antenna spacing and insertion depth.

The distribution of power and temperature generated by 915 MHz interstitial microwave antenna arrays was studied in static muscle-equivalent phantoms and both perfused and non-perfused canine thigh muscle. These arrays, which would form the geometric basis of larger volume implants, consisted of four parallel antennas oriented such that transverse to their long axes they formed the corners of a square. Arrays with 2 and 3 cm sides were compared at various depths of insertion where the nodes for all four antennas were coincident at the same depth. The position relative to the antenna nodes of the maximum power and highest temperature within the array volume varied with the depth of insertion of the antennas. Though power dropped rapidly distal to the nodes at all depths, a shift in the location of the maximum power proximal to the nodes resulted in an increase in the effective heating volume at certain insertion depths. For 2 cm array spacing the highest power and temperature were measured along the central axis of the array at all insertion depths. However, arrays using 3 cm spacing generated their maximum power adjacent to the antennas with only 50% of this level occurring along the central axis. When the temperature produced by 3 cm arrays was measured in phantoms midway through simulated 30-minute hyperthermia treatments, the effect of thermal conduction on the temperature distribution was evident. Though power was only 50% centrally, the highest temperatures occurred there. This same pattern of central heating occurred in perfused canine muscle demonstrating the importance of conductive and convective heat redistribution in reducing thermal gradients within the array volume.

Effects of replacement doses of sodium L-thyroxine on the peripheral metabolism of thyroxine and triiodothyronine in man.

Studies of the effect of L-thyroxine administration (0.3 mg daily for 7-9 wk) on the peripheral metabolism of (131)I-labeled triiodothyronine (T(3)) and (125)I-labeled thyroxine (T(4)) and on the concentration and binding of T(4) and T(3) in serum were carried out in 11 euthyroid female subjects. Administration of L-thyroxine led to consistent increases in serum T(3) concentration (137 vs. 197 ng/100 ml), T(3) distribution space (39.3 vs. 51.7 liters), T(3) clearance rate (22.9 vs. 30.6 liters/day) and absolute T(3) disposal rate (30 vs. 58 mug/day), but no change in apparent fractional turnover rate (60.3 vs. 60.6%/day). The proportion and absolute concentration of free T(3) also increased during L-thyroxine administration. Increases in serum total T(4) concentration (7.3 vs. 12.8 mug/100 ml) and in both the proportion and absolute concentration of free thyroxine also occurred. In five of the subjects, the kinetics of peripheral T(4) turnover were simultaneously determined and a consistent increase in fractional turnover rate (9.7 vs. 14.2%/day), clearance rate (0.84 vs. 1.37 liters/day), and absolute disposal rate (64.2 vs. 185.0 mug/day) occurred during L-thyroxine administration. Despite these increases in the serum concentration and daily disposal rate of both T(4) and T(3), the patients were not clinically thyrotoxic. However, basal metabolic rate (BMR) values were marginally elevated and, as in frank thyrotoxicosis, T(4)-binding capacities of thyroxine-binding globulin (TBG) and thyroxine-binding prealbumin (TBPA) reduced, suggesting that subclinical thyrotoxicosis was present. Thus, the often recommended replacement dose of 0.3 mg L-thyroxine daily may be greater than that required to achieve the euthyroid state. The studies have also provided additional evidence of the peripheral conversion of T(4) to T(3) in man and have permitted the calculation that approximately one-third of exogenously administered T(4) underwent deiodination to form T(3). To the extent that a similar fractional conversion occurs in the normal state, it can be calculated that a major fraction of the T(3) in serum derives from the peripheral deiodination of T(4) and that only a lesser fraction derives from direct secretion by the thyroid gland.

Effect of physiological variations in free fatty acid concentration on the binding of thyroxine in the serum of euthyroid and thyrotoxic subjects.

The effect of variations in the concentration of free fatty acids (FFA) on the binding of thyroid hormones in serum has been studied in 20 euthyroid subjects and 19 thyrotoxic patients. In the euthyroid group, neither the pronounced decreases in FFA induced by the oral administration of glucose or the intravenous administration of nicotinic acid, nor the marked increases in FFA which followed the administration of nicotinic acid or 2-deoxyglucose were accompanied by significant changes in the per cent of free thyroxine (T(4)), the protein-bound iodine (PBI), the per cent of endogenous T(4) bound by the T(4)-binding globulin (TBG) or T(4)-binding prealbumin (TBPA), or the resin sponge uptake of triiodothyronine (T(3)). In the thyrotoxic group, the decline in FFA concentration which followed glucose administration was accompanied by slight, but statistically significant, decreases in the PBI and both the per cent and absolute concentration of free T(4). Such changes might have been indicative of an increased intensity of T(4) binding secondary to the decrease in FFA. The serum PBI was decreased, however, a change contrary to that which would be expected to follow an increase in the intensity of T(4) binding. Furthermore, comparable changes in free T(4) and PBI did not accompany the decrease in FFA induced by the administration of insulin. Neither manipulation significantly affected the protein binding of endogenous T(4) or the resin sponge uptake of T(3). It is concluded that within a wide physiological range of concentration, FFA do not significantly influence the transport of T(4) in the serum of euthyroid subjects. In the serum of patients with thyrotoxicosis, FFA may have a slight effect on the binding of T(4), but the nature of any such effect is obscure, since parallel, rather than contrary changes in PBI and the proportion of free T(4) followed alterations in FFA concentration.

Thyroid hormone transport in the serum of patients with thyrotoxic Graves' disease before and after treatment.

Multiple indices of thyroid hormone binding have been studied in sera obtained from patients with thyrotoxic Graves' disease, before and after treatment, and in sera from a group of carefully matched normal controls. Specimens from patients with thyrotoxicosis displayed a decrease in the thyroxine (T(4))-binding capacities of T(4)-binding globulin (TBG) and T(4)-binding prealbumin (TBPA), an increase in serum protein-bound iodine (PBI), and an increase in both the proportion and absolute concentration of free T(4). In addition, a smaller than normal proportion of (131)I-labeled T(3) was associated with TBG during filter paper electrophoresis. After treatment of thyroxicosis, the only residual abnormality detected was a very slight persistent decrease in the T(4)-binding capacity of TBPA, which did not appear to influence the overall thyroid hormone-plasma protein interaction significantly, regardless of whether this was assessed under basal conditions or after enrichment of specimens with stable T(4). It is concluded that the persistent abnormalities in the peripheral metabolism of T(4), previously reported to occur in some patients with treated Graves' disease, probably do not stem from residual abnormalities in the transport of T(4) in the plasma but must arise from abnormalities in T(4) accumulation or metabolism within the tissues themselves.