Contouring workload in adjuvant breast cancer radiotherapy.

PURPOSE: To measure the impact of contouring on worktime in the adjuvant radiation treatment of breast cancer, and to identify factors that might affect the measurements. MATERIAL AND METHODS: The dates and times of contouring clinical target volumes and organs at risk were recorded by a senior and by two junior radiation oncologists. Outcome measurements were contour times and the time from start to approval. The factors evaluated were patient age, type of surgery, radiation targets and setup, operator, planning station, part of the day and day of the week on which the contouring started. The Welch test was used to comparatively assess the measurements. RESULTS: Two hundred and three cases were included in the analysis. The mean contour time per patient was 34minutes for a mean of 4.72 structures, with a mean of 7.1minutes per structure. The clinical target volume and organs at risk times did not differ significantly. The mean time from start to approval per patient was 29.4hours. Factors significantly associated with longer contour times were breast-conserving surgery (P=0.026), prone setup (P=0.002), junior operator (P<0.0001), Pinnacle planning station (P=0.026), contouring start in the morning (P=0.001), and contouring start by the end of the week (P<0.0001). Factors significantly associated with time from start to approval were age (P=0.038), junior operator (P<0.0001), planning station (P=0.016), and contouring start by the end of the week (P=0.004). CONCLUSION: Contouring is a time-consuming process. Each delineated structure influences worktime, and many factors may be targeted for optimization of the workflow. These preliminary data will serve as basis for future prospective studies to determine how to establish a cost-effective solution.

Inactivating mutations in GNA13 and RHOA in Burkitt's lymphoma and diffuse large B-cell lymphoma: a tumor suppressor function for the Gα13/RhoA axis in B cells.

G proteins and their cognate G protein-coupled receptors (GPCRs) function as critical signal transduction molecules that regulate cell survival, proliferation, motility and differentiation. The aberrant expression and/or function of these molecules have been linked to the growth, progression and metastasis of various cancers. As such, the analysis of mutations in the genes encoding GPCRs, G proteins and their downstream targets provides important clues regarding how these signaling cascades contribute to malignancy. Recent genome-wide sequencing efforts have unveiled the presence of frequent mutations in GNA13, the gene encoding the G protein Gα13, in Burkitt's lymphoma and diffuse large B-cell lymphoma (DLBCL). We found that mutations in the downstream target of Gα13, RhoA, are also present in Burkitt's lymphoma and DLBCL. By multiple complementary approaches, we now show that that these cancer-specific GNA13 and RHOA mutations are inhibitory in nature, and that the expression of wild-type Gα13 in B-cell lymphoma cells with mutant GNA13 has limited impact in vitro but results in a remarkable growth inhibition in vivo. Thus, although Gα13 and RhoA activity has previously been linked to cellular transformation and metastatic potential of epithelial cancers, our findings support a tumor suppressive role for Gα13 and RhoA in Burkitt's lymphoma and DLBCL.

The Swiss IMRT dosimetry intercomparison using a thorax phantom.

PURPOSE: In 2008, a national intensity modulated radiation therapy (IMRT) dosimetry intercomparison was carried out for all 23 radiation oncology institutions in Switzerland. It was the aim to check the treatment chain focused on the planning, dose calculation, and irradiation process. METHODS: A thorax phantom with inhomogeneities was used, in which thermoluminescence dosimeter (TLD) and ionization chamber measurements were performed. Additionally, absolute dosimetry of the applied beams has been checked. Altogether, 30 plan-measurement combinations have been used in the comparison study. The results have been grouped according to dose calculation algorithms, classified as "type a" or "type b," as proposed by Kntis et al. ["Comparison of dose calculation algorithms for treatment planning in external photon beam therapy for clinical situations," Phys. Med. Biol. 51, 5785-5807 (2006)]. RESULTS: Absolute dosimetry check under standard conditions: The mean ratio between the dose derived from the single field measurement and the stated dose, calculated with the treatment planning system, was 1.007 +/- 0.010 for the ionization chamber and 1.002 +/- 0.014 (mean+/- standard deviation) for the TLD measurements. IMRT Plan Check: In the lung tissue of the planning target volume, a significantly better agreement between measurements (TLD, ionization chamber) and calculations is shown for type b algorithms than for type a (p <0.001). In regions outside the lungs, the absolute differences between TLD measured and stated dose values, relative to the prescribed dose, [(Dm-Ds)/Dprescribed], are 1.9 +/- 0.4% and 1.4 +/- 0.3%, respectively. These data show the same degree of accuracy between the two algorithm types if low-density medium is not present. CONCLUSIONS: The results demonstrate that the performed intercomparison is feasible and confirm the calculation accuracies of type a and type b algorithms in a water equivalent and low-density environment. It is now planned to offer the intercomparison on a regular basis to all Swiss institutions using IMRT techniques.

Open low-field magnetic resonance imaging for target definition, dose calculations and set-up verification during three-dimensional CRT for glioblastoma multiforme.

AIMS: To assess the effect on target delineation of using magnetic resonance simulation for planning of glioblastoma multiforme (GBM). Dose calculations derived from computed tomography- and magnetic resonance-derived plans were computed. The accuracy of set-up verification using magnetic resonance imaging (MRI)-based digital reconstructed radiographs (DRRs) was assessed. MATERIALS AND METHODS: Ten patients with GBM were simulated using computed tomography and MRI. MRI was acquired with a low-field (0.23 T) MRI unit (SimMRI). Gross tumour volumes (GTVs) were delineated by two radiation oncologists on computed tomography and MRI. In total, 30 plans were generated using both the computed tomography, with (planbathoCT) and without (planCT) heterogeneity correction, and MRI data sets (planSimMRI). The minimum dose delivered (Dmin) to the GTV between computed tomography- and MRI-based plans was compared. The accuracy of set-up positioning using MRI DRRs was assessed by four radiation oncologists. RESULTS: The mean GTVs delineated on computed tomography were significantly (P<0.001) larger than those contoured on MRI. The mean (+/-standard deviation) Dmin difference percentage was 0.3+/-0.8, 0.1+/-0.6 and -0.2+/-1.0% for the planCT/planbathoCT-, planCT/planSimMRI- and planbathoCT/planSimMRI-derived plans, respectively. The set-up differences observed with the computed tomography and MRI DRRs ranged from 1.0 to 4.0 mm (mean 1.5 mm; standard deviation+/-1.4). CONCLUSIONS: GTVs defined on computed tomography were significantly larger than those delineated on MRI. Compared with computed tomography-derived plans, MRI-based dose calculations were accurate. The precision of set-up verifications based on computed tomography- and MRI-derived DRRs seemed similar. The use of MRI only for the planning of GBM should be further assessed.

LTB4-induced transient neutropenia in the rat: a model for evaluating efficacy and bioavailability of LTB4 receptor antagonists.

An animal model of leukotriene B4- (LTB4) induced neutropenia has been developed to evaluate LTB4 receptor antagonists in vivo. LTB4, a potent chemotactic inflammatory mediator, when administered intravenously, induces a profound, rapid, and transient redistribution of blood neutrophils from the circulating pool to the marginated pool. This phenomenon is applied in the neutropenia model whereby circulating blood neutrophil counts prior to and after intravenous infusion of LTB4 are compared. Kinetics of LTB4-induced neutrophil responses are determined through the use of a Technicon H*1 automated blood cell analyzer. LTB4 receptor antagonists are identified by inhibition of LTB4-induced neutropenia. Standard antiinflammatory compounds including BW-755C, Abbott A-64077 (zileuton), dexamethasone-21-acetate, indomethacin, and naproxen did not affect LTB4-induced neutropenia. A potent LTB4 receptor antagonist, designated "RPR," inhibited LTB4-induced neutropenia following oral administration in a dose-dependent fashion.

Magnetic resonance imaging and morphometric quantitation of cartilage histology after chronic infusion of interleukin 1 in rabbit knees.

Cartilage pathology in rabbit knees was monitored by noninvasive magnetic resonance imaging (MRI) and evaluated using morphometric histologic measurements. Infusion of rabbit knees with the cytokine interleukin 1 induces cartilage degradation and inflammation. A miniosmotic pump was implanted subcutaneously to deliver interleukin 1 through a polyethylene catheter inserted into the rabbit knee. Rabbit knees were imaged using MRI and prepared for histologic examination at 5 and 12 days after chronic infusion of interleukin 1. MRI obtained 0.7-mm sections for three-dimensional reconstruction of cartilage image. Cartilage deterioration near the site of infusion was visible on MRI. MRI measurements indicated a reduction in cartilage thickness. Histology revealed a loss of staining of cartilage matrix proteoglycan, synovial hypertrophy, and perichondral bone resorption. Morphometric analysis of cartilage histology indicated a reduction in both cellularity (chondrocytes/m mu 2 area) and cell to matrix area ratio. These observations suggest that a loss of proteoglycan, an early event in cartilage degeneration, can be detected by MRI.

Direct evidence for active metalloproteinases mediating matrix degradation in interleukin 1-stimulated human articular cartilage.

When adult human articular cartilage was maintained in organ culture in the presence of interleukin 1 beta, increased destruction of the extracellular matrix was observed, as judged by increased type II collagen degradation in situ determined immunohistochemically and the increased release of proteoglycan into the culture medium. Concomitant with these changes was the increased release of latent metalloproteinases into the culture medium. Culture of cartilage in the presence of a peptidylhydroxamate metalloproteinase inhibitor indicated a key role for the active forms of these enzymes in situ, since it produced a marked reduction in both proteoglycan release and collagen degradation. This compound had no detectable cytotoxic effects in organ culture and did not reduce the secretion of the metalloproteinases. The results of this study provide direct evidence that the latent metalloproteinase precursors, whose release is greatly stimulated by interleukin 1, are indeed activated to some degree and participate in cartilage matrix degradation.

Comparison of magnetic resonance imaging (MRI) and histopathology in rabbit models of osteoarthritis and immune arthritis.

Osteoarthritis was surgically induced in mature male Dutch Belted rabbits by sectioning the fibular collateral and sesamoid ligaments and removal of the anterior horn of the lateral meniscus. The site of surgical intervention was detectable by MRI. Histopathologic analysis revealed severe focal cartilage lesions on opposing surfaces of the tibia and femur. Histology of cartilage adjacent to the osteoarthritic lesions appeared normal. In another animal model, arthritis was induced by immunization against ovalbumin followed by intra-articular injection of ovalbumin. MRI of immune arthritic rabbit knees showed accumulation of synovial fluid and cartilage degradation. Histopathology was characterized by vascular necrosis of the synovium and depletion of cartilage proteoglycan. MRI can be used to non-invasively follow the therapeutic effects of drug treatment on synovial inflammation and cartilage degradation in rabbit knees.

Pathogenesis of Lactobacillus casei-induced polyarthritis in Lewis rats: 1. Time related changes in histopathological scores and hematology.

Intraperitoneal injection of cell wall fragments from L. casei (ATCC 11578) induces an acute and a chronic inflammatory arthritis of the distal joints of LEW/N female rats. Histopathological changes in four distal joints and hematologic changes were analyzed on days 3, 10, 20, 30, 40, 50 and 59. All joints were scored for changes in inflammation, pannus, cartilage and bone. The acute inflammatory response consisted of fluid exudate, fibrin, neutrophils and some macrophages concentrated along the periosteum of the longer bones. The disease progressed with synovial fibroblast proliferation and infiltration of lymphocytes and macrophages. On day 10, cartilage changes were associated with pannus formation and subchondral fibrosis. Both localized bone resorption and periosteal new bone formation were features of the chronic phase. Lymphocytes were elevated above normal (p < 0.05) on day 3, 10, 20, 30 and 40; returning to the normal range on day 50 and 59. Neutrophils were elevated on days 10, 20, 30, 40 and 59. L. casei-induced polyarthritis in Lewis rats appears to be a fibroblast-, macrophage-mediated disease with a prominent lymphoid component.

Pathogenesis of Lactobacillus casei-induced polyarthritis in Lewis rats: 2. Time related changes in organ weights and liver enzymes.

Hepatic enzymes and organ weights were measured in LEW/N female rats during the acute and the chronic phases of L. casei-induced arthritis on day 3 and days 30 and 59, respectively. In the acute phase, day 3, adrenal and spleen weights were increased and thymus weights were decreased in L. casei arthritic rats as compared to normal control rats. Adrenal, liver, kidney, spleen and thymus weights of arthritic rats were in the normal range on days 30 and 59. Liver cytochrome P450, aminopyrine N-demethylase and analine hydroxylase were reduced in livers of L. casei-treated rats on day 3 as compared to normal controls. On days 30 and 59 hepatic enzymes in L. casei-arthritic rats were in the normal range. Unlike adjuvant arthritis in which changes in liver enzymes alter drug metabolism; after the acute onset of L. casei-induced arthritis, hepatic enzymes return to the normal range.

Effect of 5-lipoxygenase inhibitors on in situ LTB4 biosynthesis following calcium ionophore stimulation in the rat pleural cavity.

The intrapleural injection of carrageenan in the rat induces exudate formation, cellular influx and leukotriene generation in the pleural cavity. We have demonstrated that the inflammatory response (exudate volume, and LTB4 levels) is increased in situ by the intrapleural administration of calcium ionophore A 23187 (100 nmol/rat) at 4, 16, 24, 48, and 72 h after the injection of carrageenan and that the A 23187-induced increase is dose-dependent. The oral administration of A-64077 and MK-886, two 5-lipoxygenase inhibitors (5-LOIs), at 10 mg/kg causes marked decreases in LTB4 release at the above-mentioned time intervals. However, A 23187-induced augmented exudate formation is not affected by the treatment with 5-LOIs. The results suggest that the use of 5-LOIs to inhibit LTB4 biosynthesis may be beneficial in various LTB4-dependent pathological conditions.

Effect of anti-inflammatory compounds on edema formation and myeloperoxidase activity in the arachidonic acid-induced ear model in the mouse.

The arachidonic acid (AA)-induced ear edema model in the mouse has been demonstrated as an effective in vivo experimental tool to screen compounds showing anti-inflammatory activity. Since neutrophil influx is a component of the inflammatory reaction, we have modified this assay by quantitating myeloperoxidase (MPO) levels which reflect neutrophil accumulation in the edematous biopsies of the mouse ear. Our work has shown that orally administered 5-lipoxygenase inhibitors, dual inhibitors (CO/LO), and steroids dose-dependently inhibit both edema formation and MPO activity, whereas oral activity is not seen with NSAID's. There is a good correlation between the inhibition of edema formation and of MPO activity by these compounds. Thus, measurement of MPO, in addition to the AA-induced edema in the mouse ear, can provide another parameter to profile potential anti-inflammatory compounds.

Pharmacological modulation of rat monocytes: in vivo effects on Ia expression and interleukin-1 production.

We have shown that during the developing phase of adjuvant disease (AD) in rats the expression of MHC class II (Ia) antigens on blood monocytes (BM) was enhanced. The results of a study in established AD are reported now. Four agents were tested: indomethacin and diclofenac-sodium (1 mg/kg/day); levamisole and prinomide (10 mg/kg/day), administered orally from day 18-31 after induction of AD. We assessed the following BM parameters: Ia expression, interleukin-1 (sIL-1) production, and membrane bound IL-1 (mIL-1). In AD Ia expression was enhanced, no changes occurred in mIL-1 or sIL-1. Indomethacin treatment reduced sIL-1 production, levamisole Ia expression and mIL-1 activity, prinomide all three parameters measured and diclofenac, though clinically effective, none.

Degradation of rat chondrosarcoma proteoglycans by a neutral metalloprotease from rabbit chondrocytes.

Rat chondrosarcoma proteoglycan aggregate with radiolabeled core protein was digested with a chondrocyte metalloprotease (CMP) or clostripain (CP) at neutral pH. The rates of product formation and the sizes and antigenicities of the products were studied using column chromatography and monoclonal antibodies. Sixteen percent of [35S]methionine label and 17-18% of [3H]serine label in core protein were freed from glycosaminoglycan bound peptides by 50 U/ml (760 micrograms/ml) of CP or 10 micrograms/ml (estimated) of CMP in 180 min. The CP reaction was almost complete at 5 minutes while the CMP reaction proceeded slowly from 5 to 180 min. The chondroitin-sulfate rich fragments were smaller after CP than CMP treatment. The 180 min CMP digest contained protein that migrated in 2 peaks on Sepharose CL6B. These two peaks corresponded to the peaks where hyaluronic acid binding region produced by CP and link protein migrate. Metalloenzyme inhibitors inhibited CMP with IC50s of 5 x 10(-5)M, 1 x 10(-3)M, and 80 micrograms/ml for phenanthroline, EDTA, and alpha 2-macroglobulin, respectively.

Protease inhibitors decrease rabbit cartilage degradation after meniscectomy.

In vitro proteoglycan (PG) synthesis and release were measured on cartilage removed from rabbit knees within 1 week of meniscectomy. Three days following partial lateral meniscectomy, 72% of the femurs and 82% of the tibias had visible ulcers. Cartilage from the weight-bearing areas incorporated 2.0-2.9 times more 35S-sulfate in vitro than cartilage from the opposite, unoperated knees. 3H-thymidine incorporation was 2.5-3.4 times higher for surgical than control groups. 35S-sulfate incorporation by the surgical group was inhibited by 22% in the presence of 10(-4) M U24522, an inhibitor of rabbit chondrocyte metalloprotease (CMP). 3H-thymidine incorporation by the surgical group was inhibited by 28% by 10(-4) M U24522. In vitro PG release from cartilage removed 2 days after surgery was 1.6-3.7 times higher for the surgical than the control group. PG release by the surgical group after 22 h of incubation was reduced to the control level by three CMP inhibitors, U24278, U24279, and U24522. PG release by cartilage from the nonsurgical group was also reduced by these compounds at 22 h. These results suggest that both the anabolic and catabolic processes that are stimulated by surgery can be isolated in vitro and that CMP may be involved in the catabolic process.

The potential use of implanted radiolabelled bovine nasal cartilage in dialysis tubing to evaluate agents affecting cartilage degradation in vivo.

Cartilage which undergoes extensive autolysis in vitro (spontaneous or stimulated) is characterized by proteoglycan loss. Experimental conditions and inhibitor profils studies suggest neutral metalloproteinases induce the autolysis. In these preliminary studies we compared the degradation of Na2 35SO4 labeled bovine nasal cartilage (BNC) plugs placed in dialysis tubing in vitro and in vivo. The dialysis tubing was used to exclude large molecules (molecular weights greater than 2000) like proteinases, and proteinase inhibitors (e.g. alpha 2-macroglobulin) but not potential test agents from the implanted cartilage. Cartilage autolysis occurred with live tissue but not with heat-killed tissue in both the in vitro and in vivo systems. In addition retinoic acid and phenanthroline were effective when placed inside or outside the dialysis tubing. A potentially useful procedure to evaluate agents which affect cartilage degradation is described.

Interleukin-1 induces chondrocyte protease production: the development of collagenase inhibitors.

Supernatants from the P388D1 macrophage cell line as well as human interleukin-1 (IL-1) stimulated primary rabbit articular chondrocytes to produce collagen- (C-ase) and proteoglycan- (PG-ase) degrading proteases. The P388D1 derived factor had a molecular weight of 16,000-20,000 and a pI of 4.5-5.0. Both protease activities were metal dependent and inhibited by EDTA, phenanthroline, and alpha 2-macroglobulin but not by PMSF, TLCK, pepstatin, or alpha 1-antitrypsin. Size exclusion chromatography indicated the molecular weights for latent PG-ase and C-ase were 44,000-56,000 and 34,000-44,000, respectively. Chemical synthesis efforts produced two classes of C-ase inhibitors--thiols and hydroxamic acids. The former had IC50 values of 10(-5)-10(-6) M while the latter approached 10(-7) M.

Proteoglycan degradation by a chondrocyte metalloprotease. Effects of synthetic protease inhibitors.

Synthetic inhibitors of a chondrocyte metalloprotease (CMP) were assessed for potency. Proteoglycan core protein was used as substrate. The IC50 values were between 2 X 10(-6) and 7 X 10(-6) M for two types of inhibitors, thiol tripeptides and N-carboxyalkyl peptides. Hydroxamic acid peptides were more potent, with IC50 values of 3.2 X 10(-8) to 6.0 X 10(-8) M. These results confirm inhibitory concentrations reported using a proteoglycan-polyacrylamide bead assay. The slopes of the dose-response curves for the thiol compounds were steeper than the slopes for the other two types of compounds. All of the culture media tested inhibited CMP to some extent. Some media also interfered with inhibitor activity. In Ham's F10 nutrient medium, minimum CMP inhibition occurred, and all four hydroxamic acid peptides retained their activity for 1-2 days at 37 degrees. One thiol peptide compound assayed lost activity in 1 hr in thiocyanate-treated serum. All four hydroxamic acid peptides assayed retained activity in thiocyanate-treated serum after 3 days at 37 degrees. The hydroxamic acid peptides may provide a way to block endogenous CMP activity in vivo and to assess the role of CMP in normal and experimentally altered cartilage. They are more potent than other known CMP inhibitors. They retain activity in culture media and serum conditions used for in vivo and in vitro tests of CMP activity and toxicity.

Effect of synthetic metalloprotease inhibitors on cartilage autolysis in vitro.

The role of chondrocyte metalloprotease (CMP) in mediating cartilage autolysis was studied. Proteoglycan (PG) release and synthesis by rabbit articular cartilage explants were measured. After a 1-day preculture in control medium, 3.3 X 10(-6) M retinoic acid (RET) treatment for 1 day stimulated PG release several fold. RET also caused a large decrease in PG synthesis that returned to the control level after a 3-day recovery period. The effect on PG synthesis was observed at serum levels of 5 and 0.05%. The effect of RET on PG release required protein synthesis, inasmuch as it was lost in cultures maintained in media without amino acids or in a low volume of media. Interleukin-1 (IL-1) and lipopolysaccharide (LPS) treatment for 2 days also stimulated PG release. More PG was released after RET than after IL-1 or LPS, and only RET produced an effect that was evident by day 1. The amount of CMP that produced the same size effect on PG release as these stimulators was below the detection level of PG protease assays. Three potent CMP inhibitors reduced RET-, IL-1- and LPS-stimulated PG release to control levels. These inhibitors did not block another action of RET on chondrocytes, namely the inhibition of PG synthesis by RET immediately after treatment. The inhibitors did not act by reducing cell viability, because recovery of the rate of PG synthesis 3 days post-treatment occurred in inhibitor-treated cultures. These studies suggest that CMP is involved in cartilage autolysis that is stimulated by RET and IL-1.