Dose constraints for whole breast radiation therapy based on the quality assessment of treatment plans in the randomised Danish breast cancer group (DBCG) HYPO trial.

PURPOSE: Quality assessment of the treatment plans in the Danish Breast Cancer Group (DBCG) HYPO trial was carried out based on prospectively reported dosimetric parameters and evidence-based dose constraints for whole breast radiation therapy were derived. MATERIALS AND METHODS: From 2009 to 2014, 1882 patients (pts) were randomised between 50 Gy/25fractions (fr) versus 40 Gy/15fr. Doses to CTVp_breast (V95%, V107%-V110%, Dmax, and in addition for 40 Gy plans V105%-V107%), ipsilateral lung (V20Gy/V17Gy), heart (V20Gy/V17Gy, V40Gy/V35Gy), and left anterior descending coronary artery (LADCA) (Dmax) and use of respiratory gated technique were prospectively reported to the DBCG database. After end of accrual, these dosimetric parameters from all plans in the trial were compared to the pre-specified treatment constraints. RESULTS: In total, 1854 pts from eight radiation therapy (RT) centres in three countries were treated. No statistically significant differences were found between the results for 40 Gy and 50 Gy plans, except for CTVp_breast hot-spot volume (V107%-V110%). Of the 40 Gy pts, 90% with CTVp_breast > 600 mL and 95% with CTVp_breast ≤ 600 mL had a CTVp_breast hot-spot volume (V105%-V107%) <2%. In 95% of the 50 Gy plans, the CTVp_breast absolute hot-spot volume (V107%-V110%) was <0.5 mL and 1.7 mL for CTVp_breast ≤ 600 mL and > 600 mL, respectively. Compliance was >99% for both heart and lung constraints. Largest deviation from protocol constraints was found for the volume of CTVp_breast covered with 95% of the prescription dose or more (V95%). The CTV dose coverage (V95%) was >94.3% in 95% of the right-sided pts, whereas the figures for 95% of the left-sided pts treated with and without respiratory gating were 93.2% and 88.8%, respectively. CONCLUSION: A high degree of compliance with protocol dose constraints was found for treatment plans in the DBCG HYPO trial. New constraints for dose to organs at risk and high-dose volumes in the breast are suggested for breast-only RT planning.

Effect of the pyrrolopyrimidine lipid peroxidation inhibitor U-101033E on neuronal and astrocytic metabolism and infarct volume in rats with transient middle cerebral artery occlusion.

The aim of the present study was to assess the effect of post ictal administration of the pyrrolopyrimidine lipid peroxidation inhibitor, U-101033E, on infarct volume and neuronal and astrocytic metabolism in rats with transient middle cerebral artery occlusion (MCAO). Rats were subjected to 120 min of MCAO followed by 140 min of reperfusion and randomly assigned to control (n=17) or U-101033E treatment (n=16). Drug infusion started 5 min after MCAO and lasted 220 min with a 15 min interruption during the reperfusion procedure. Sixteen rats underwent diffusion weighted imaging 260 min after ictus, from which the apparent diffusion coefficient (ADC) was determined. Seventeen rats received an iv bolus injection of [1-13C]glucose and [1,2-13C]acetate 245 min after ictus. Tissue extracts from two brain regions representing penumbra and ischemic core were analyzed with 13C NMRS and HPLC. U-101033E did not affect the volume of ischemic tissue estimated from the ADC maps. In the penumbra, U-101033E specifically decreased mitochondrial pyruvate metabolism via both pyruvate dehydrogenase and pyruvate carboxylase pathways. Thus, U-101033E impaired both neuronal and astrocytic mitochondrial pyruvate metabolism. At the same time anaerobic glucose usage was increased, leading to increased lactate labeling and content. Also alanine labeling was increased. The data do not support lactate as an important substrate for neuronal mitochondria in ischemia-reperfusion. A similar pattern of reduced mitochondrial pyruvate metabolism and increased cytosolic pyruvate metabolism was found in the irreversibly damaged ischemic core. The present study highlights the importance of other outcome measures than ischemic tissue volume for evaluation of drug efficacy in animal models, which in turn could increase the likelihood of success in clinical trials.

Hyperbaric oxygen and neutrophil accumulation/tissue damage during permanent focal cerebral ischaemia in rats.

The use of hyperbaric oxygenation (HBO) for the treatment of severe brain ischaemia remains controversial. The HBO may interfere with destructive neutrophil (PMN) infiltration following ischaemia/reperfusion. The effects of HBO on PMN accumulation and the area of ischaemic tissue damage were investigated in rats having permanent focal ischaemia (4 h). The right middle cerebral arteries of a group of Wistar rats were permanently occluded. The rats were then randomly divided into those ( n=7) to be treated with HBO at 2 atm for 230 min and those ( n=8) to breathe air at atmospheric pressure for an equivalent period. The HBO had no effect on permanent ischaemia, as there was no significant difference in the area of ischaemic tissue damage between HBO-treated [mean (SD)] [331 (88) mm(3)] and non-treated animals [322 (111) mm(3)]. Moreover, the increase in myeloperoxidase [5.4 (4.1) compared to 2.4 (1.2) pg x g(-1) wet weight of brain] was not significantly different. The results indicate that HBO did not reduce tissue damage during 4 h of permanent focal ischaemia.

Local endostatin treatment of gliomas administered by microencapsulated producer cells.

We describe a technique for the treatment of malignant brain tumors based on local delivery of the anti-angiogenic protein endostatin from genetically engineered cells encapsulated in ultrapure sodium alginate. Alginate consists of L-guluronic and D-mannuronic acid, which in the presence of divalent cations forms an extended gel network, in which cells reside and remain immunoisolated, when implanted into the rat brain. Here, we show that endostatin-transfected cells encapsulated in alginate maintain endostatin secretion for at least four months after intracerebral implantation in rats. During the implantation period 70% of the encapsulated cells remained viable, as opposed to 85% in in vitro-cultured capsules. Rats that received transplants of BT4C glioma cells, together with endostatin-producing capsules (0.2 microg/ml per capsule), survived 84% longer than the controls. The endostatin released from the capsules led to an induction of apoptosis, hypoxia, and large necrotic avascular areas within 77% of the treated tumors, whereas all the controls were negative. The encapsulation technique may be used for many different cell lines engineered to potentially interfere with the complex microenvironment in which tumor and normal cells reside. The present work may thus provide the basis for new therapeutic approaches toward brain tumors.

Hyaluronidase-induced periodic modulation of the interstitial fluid pressure increases selective antibody uptake in human osteosarcoma xenografts.

Periodic modulation of the elevated interstitial fluid pressure might improve filtration and uptake of tumor-targeting macromolecules (e.g. radioimmunoconjugates) in solid tumors. Cycling of the tumor interstitial fluid pressure was initiated by intratumoral injections of bovine testicular hyaluronidase (BTH, 1,600 U) in osteosarcoma-bearing nude mice. BTH injection was repeated at 3-day intervals up to 9 days, in conjunction with tail vein injections of 125I-labeled TP-3 monoclonal antibody against an osteosarcoma-associated antigen (n = 9) or non-specific 125I-labeled UPC-10 antibody (n = 9). Control mice received intratumoral injections of phosphate buffered saline (n = 18). The radioactivities of tumor and normal tissues (blood, liver, kidney and spleen) were measured and compared between the different groups. BTH injections increased the tumor uptake of specific 125I-labeled TP-3 significantly by approximately 70% in mice receiving 3 fractions compared to 1-2 fractions of the antibody (p < 0.05). The tumor/normal tissue ratio in mice receiving 3 fractions of 125I-labeled TP-3 (n = 5) was significantly higher for all tissues, compared with mice receiving 1-2 fractions (n = 4) (p < 0.05). Control injections did not affect the tumor/blood ratio, but increased the uptake of 125I-labeled TP-3 significantly in kidney and spleen (p < 0.05). Also, BTH reduced the uptake of 125I-labeled UPC-10 in tumor and liver by approximately 20% compared with controls (p < 0.05). The results indicate that periodic lowering of the tumor interstitial fluid pressure might increase the specificity of blood-borne monoclonal antibodies to solid tumors in vivo.

Uptake, penetration, and binding of monoclonal antibodies with increasing affinity in human osteosarcoma multicell spheroids.

BACKGROUND: Multicell spheroids from the human osteosarcoma cell line, OHS, were incubated with increasing concentrations of the monoclonal antibodies TP-1, TP-3 and 9.2.27 having different affinities (Ka = 8.5 x 10(8) M-1, 3.4 x 10(9) M-1 and 1.4 x 10(11) M-1, respectively). MATERIALS AND METHODS: Uptake and penetration of the fluorescein labelled antibodies were studied using confocal laser scanning fluorescence microscopy, and antibodies bound per cell were measured using flow cytometry. RESULTS: The antibody with highest affinity, 9.2.27, bound to all available binding sites in one cell-layer before reaching the next, whereas TP-3 and TP-1 gradually bound to an increasing number of epitopes of all cells throughout the spheroids. The penetration rate and antibody uptake increased with increasing antibody concentration up to saturating concentrations. 9.2.27 required saturation concentrations to reach the center of the spheroids, whereas TP-1 and TP-3 penetrated the centre when concentrations below saturation were applied. CONCLUSION: The antibodies might be useful in radioimmunotherapy of micrometastases.

Neuroprotective effect of the novel glutamate AMPA receptor antagonist YM872 assessed with in vivo MR imaging of rat MCA occlusion.

The neuroprotective effect of post-ischemic treatment with the novel, highly water-soluble, glutamate AMPA receptor antagonist YM872 was evaluated by using MR imaging and histopathology of rats subjected to permanent MCA occlusion. Two treatment groups with continuous i.v. infusion of 20 mg kg-1 h-1 YM872 during either the first 4 h or first 24 h after MCA occlusion, called 4 h YM872 treatment group (n=9) and 24 h YM872 treatment group (n=8) respectively, were compared to a control group (n=8). The main end-point was T2 weighted MR imaging and histopathology 24 h after MCA occlusion. Also the time evolution of the ischemic tissue damage was studied by diffusion weighted MR imaging 412 and 24 h after MCA occlusion. The volume of ischemic tissue damage as assessed by diffusion weighted MR imaging 412 h after MCA occlusion was significantly smaller in both YM872 treatment groups (99+/-52 mm3 and 102+/-44 mm3 compared to 186+/-72 mm3 in the control group, +/-S.D. and p=0.008). The infarct volume as assessed by T2 weighted MR imaging 24 h after MCA occlusion was significantly smaller only in the 24 h YM872 treatment group (262+/-57 mm3 compared to 366+/-49 mm3 in the control group, +/-S.D. and p=0.01) while the infarct volume in the 4 h YM872 treatment group (357+/-88 mm3) was similar to the control group. YM872 treatment significantly reduced the infarct volume 24 h after MCA occlusion when the drug was administered as continuous infusion during the 24-h observation period.

Comparison of extracellular matrix in human osteosarcomas and melanomas growing as xenografts, multicellular spheroids, and monolayer cultures.

BACKGROUND: The composition of extracellular matrix in human xenografts and spheroids were compared with the monolayer cultures from which they originated. Collagen I, fibronectin, acetylglucosamine, and acetylgalactosamine were quantitated in two osteosarcomas and one melanoma. METHODS: Using fluorescence microscopy, extracellular matrix constituents in the cellular and extracellular compartment were measured, whereas flow cytometry measured the extracellular matrix constituents bound to the cell surface as well as the total cellular amount including intracellular and surface bound constituents. RESULTS: The fluorescence microscopy measurements, demonstrated that the xenografts contained more or equal quantities of the extracellular matrix constituents compared with the spheroids. Flow cytometric measurements of total cellular amounts, showed that cells from xenografts usually contained more or equal amounts as the spheroid cells, which contained less or equal amounts as the monolayer cells. The surface expression of the extracellular matrix constituents increased or there were no significant differences, comparing cells grown as monolayers, spheroids, and xenografts. CONCLUSIONS: The data shows that multicellular spheroids being an in vitro system of intermediate complexity between monolayer cultures and tumours, contain an extracellular matrix corresponding to some degree to this intermediate position.

Penetration and binding of monoclonal antibody in human osteosarcoma multicell spheroids. Comparison of confocal laser scanning microscopy and autoradiography.

Penetration and binding of monoclonal antibody (MAb) in multicell osteosarcoma spheroids have been studied by autoradiography and confocal laser scanning microscopy (CLSM). Optical sectioning of the 3-dimensional spheroids was performed by CLSM. Owing to attenuation of fluorescence intensity, FITC-labelled MAb could not be detected at depths greater than 60 microm within the spheroids. The antibody uptake seen in autoradiographs and CLSM images 60 microm within the spheroids were essentially identical. MAb had reached all parts of the spheroids within 6 h. Quantitative measurements of the fluorescence intensity of FITC-labelled MAb seen in confocal images and measurements of MAb bound per cell using flow cytometry, showed that maximum uptake was reached after 6 h. The possibility to perform both quantitative and qualitative measurements makes CLSM a promising method for studying antibody uptake in thick tissue samples.

Flow cytometric measurements of heat shock proteins.

A fixation and immunofluorescence staining procedure for measurements of heat shock proteins (hsp) by flow cytometry is reported. Three fixatives were compared: 80% methanol at -20 degrees C for 1 h, 70% ethanol at 0 degree C for 1 h, and 3% paraformaldehyde at 4 degrees C for 1 h followed by 0.2% NP-40. Cells fixed with methanol showed strongest immunofluorescence and lowest nonspecific fluorescence. The level of hsp 70 as a function of time after heating followed the same kinetics as the development of thermotolerance reported by others. The level of hsp 70 increased with increasing heat dose up to a maximum heat dose, and above this heat dose a decrease in the level of hsp 70 was observed. Correlated measurements of the level of hsp 70 and DNA showed that hsp 70 was found in all phases of the cell cycle. The level of hsp 70 increased about two-fold in unheated cells throughout the cell cycle. The increase in G2 + M cells compared with G1 cells was lower in cells heated at 45 degrees C for 20 min followed by incubation at 37 degrees C for 24 h before fixation and staining, than in unheated cells. The results show that flow cytometry provides a rapid and quantitative technique for measuring hsp. Correlated measurements of hsp and other cellular parameters might also be obtained.