Interfractional variation in whole-breast VMAT irradiation: a dosimetric study with complementary SGRT and CBCT patient setup.

BACKGROUND: The dosimetric effect of setup uncertainty and tissue deformations in left-sided whole-breast irradiation with complementary surface-guided radiotherapy (SGRT) and cone-beam computed tomography (CBCT) setup was evaluated. METHOD: Treatment courses of 40.05 Gy prescribed dose in 15 fractions were simulated for 29 patients by calculating the dose on deformed CT images, that were based on daily CBCT images, and deforming and accumulating the dose onto the planning CT image. Variability in clinical target volume (CTV) position and shape was assessed as the 95% Hausdorff distance (HD95) between the planning CTV and deformed CTV structures. DVH metrics were evaluated between the planned and simulated cumulative dose distributions using two treatment techniques: tangential volumetric modulated arc therapy (tVMAT) and conventional 3D-conformal radiotherapy (3D-CRT). RESULTS: Based on the HD95 values, the variations in CTV shape and position were enclosed by the 5 mm CTV-PTV margin in 85% of treatment fractions using complementary CBCT and SGRT setup. A residual error of 8.6 mm was observed between the initial SGRT setup and CBCT setup. The median CTV V95% coverage was 98.1% (range 93.1-99.8%) with tVMAT and 98.2% (range 84.5-99.7%) with 3D-CRT techniques with CBCT setup. With the initial SGRT-only setup, the corresponding coverages were 96.3% (range 92.6-99.4%) and 96.6% (range 84.2-99.4%), respectively. However, a considerable bias in vertical residual error between initial SGRT setup and CBCT setup was observed. Clinically relevant changes between the planned and cumulative doses to organs-at-risk (OARs) were not observed. CONCLUSIONS: The CTV-to-PTV margin should not be reduced below 5 mm even with daily CBCT setup. Both tVMAT and 3D-CRT techniques were robust in terms of dose coverage to the target and OARs. Based on the shifts between setup methods, CBCT setup is recommended as a complementary method with SGRT.

Breast deformation during the course of radiotherapy: The need for an additional outer margin.

PURPOSE: The aim of this retrospective study was to investigate and quantify the extent of breast deformation during the course of breast cancer (BC) radiotherapy (RT). The magnitude of breast deformation determines the additional outer margin needed for treatment planning to deliver a full dose to the target volume. This is especially important when using inverse planning techniques. METHODS: A total of 93 BC patients treated with RT and with daily CBCT image guidance were selected for this study. Patients underwent either only breast-conserving surgery (BCS) (n = 5), BCS with sentinel node biopsy (n = 57) or BCS with radical axillary node dissection (n = 31). The treatment area included the whole breast and chest wall (54%) or also the axillary lymph nodes (46%). 3D-registration was conducted between 1731 CBCT images and the respective planning CT images to assess the difference in breast surface. RESULTS: The largest maximum breast surface expansion (MBSE) was 15 mm; the average was 2.4 ±â€¯2.1 mm. In 294 fractions (17%), the MBSE was ≥5 mm. An outer margin of 8 mm would have been required to cover the whole breast in 95% of the treated fractions. There was a statistically significant correlation between the MBSE and body mass index (r = 0.38, p = 0.001). CONCLUSIONS: Significant changes in the breast surface occur during the course of BC RT which should be considered in treatment planning. An additional margin outside the breast surface of at least 8 mm is required to take into account the anatomical changes occurring during BC RT.

Vitamin D status and correlates of low vitamin D in schizophrenia, other psychoses and non-psychotic depression - The Northern Finland Birth Cohort 1966 study.

There is limited knowledge available on the association of vitamin D with psychiatric disorders in young adults. We aimed to investigate vitamin D levels and associating factors in schizophrenia, other psychoses and non-psychotic depression. We studied 4,987 participants from the Northern Finland Birth Cohort 1966 (31 years) with available serum 25-hydroxyvitamin D [25(OH)D] measurements. The final sample was divided into four groups: schizophrenia (n = 40), other psychoses (n = 24), non-psychotic depression (n = 264) and control (n = 4659). To account for the influence of environmental and technical covariates, we generated a vitamin D score variable with correction for season, sex, batch effect and latitude. We further examined how vitamin D levels correlate with anthropometric, lifestyle, socioeconomic and psychiatric measures. Neither serum 25(OH)D concentration nor vitamin D score differed between schizophrenia, other psychoses, non-psychotic depression and control group. The prevalence of vitamin D deficiency was 3.2%, insufficiency 25.5%, and sufficiency 71.3%. Low vitamin D score correlated with regular smoking in the group with schizophrenia. No difference was observed in other psychiatric conditions. We did not find any difference in vitamin D status between schizophrenia, psychoses, non-psychotic depression and control groups, but future studies are warranted to elucidate the role of vitamin D in psychiatric conditions.

Androgen-regulated miR-32 targets BTG2 and is overexpressed in castration-resistant prostate cancer.

The androgen receptor (AR) signaling pathway is involved in the emergence of castration-resistant prostate cancer (CRPC). Here, we identified several androgen-regulated microRNAs (miRNAs) that may contribute to the development of CRPC. Seven miRNAs, miR-21, miR-32, miR-99a, miR-99b, miR-148a, miR-221 and miR-590-5p, were found to be differentially expressed in CRPC compared with benign prostate hyperplasia (BPH) according to microarray analyses. Significant growth advantage for LNCaP cells transfected with pre-miR-32 and pre-miR-148a was found. miR-32 was demonstrated to reduce apoptosis, whereas miR-148a enhanced proliferation. Androgen regulation of miR-32 and miR-148a was confirmed by androgen stimulation of the LNCaP cells followed by expression analyses. The AR-binding sites in proximity of these miRNAs were demonstrated with chromatin immunoprecipitation (ChIP). To identify target genes for the miRNAs, mRNA microarray analyses were performed with LNCaP cells transfected with pre-miR-32 and pre-miR-148a. Expression of BTG2 and PIK3IP1 was reduced in the cells transfected with pre-miR-32 and pre-miR-148a, respectively. Also, the protein expression was reduced according to western blot analysis. BTG2 and PIK3IP1 were confirmed to be targets by 3'UTR-luciferase assays. Finally, immunostainings showed a statistically significant (P<0.0001) reduction of BTG2 protein in CRPCs compared with untreated prostate cancer (PC). The lack of BTG2 staining was also associated (P<0.01) with a short progression-free time in patients who underwent prostatectomy. In conclusion, androgen-regulated miR-32 is overexpressed in CRPC, leading to reduced expression of BTG2. Thus, miR-32 is a potential marker for aggressive disease and is a putative drug target in PC.

Overexpression of androgen receptor enhances the binding of the receptor to the chromatin in prostate cancer.

Androgen receptor (AR) is overexpressed in the majority of castration-resistant prostate cancers (CRPCs). Our goal was to study the effect of AR overexpression on the chromatin binding of the receptor and to identify AR target genes that may be important in the emergence of CRPC. We have established two sublines of LNCaP prostate cancer (PC) cell line, one overexpressing AR 2-3-fold and the other 4-5-fold compared with the control cells. We used chromatin immunoprecipitation (ChIP) and deep-sequencing (seq) to identify AR-binding sites (ARBSs). We found that the number of ARBSs and the AR-binding strength were positively associated with the level of AR when cells were stimulated with low concentrations of androgens. In cells overexpressing AR, the chromatin binding of the receptor took place in 100-fold lower concentration of the ligand than in control cells. We confirmed the association of AR level and chromatin binding in two PC xenografts, one containing AR gene amplification with high AR expression, and the other with low expression. By combining the ChIP-seq and expression profiling, we identified AR target genes that are upregulated in PC. Of them, the expression of ZWINT, SKP2 (S-phase kinase-associated protein 2 (p45)) and FEN1 (flap structure-specific endonuclease 1) was demonstrated to be increased in CRPC, while the expression of SNAI2 was decreased in both PC and CRPC. FEN1 protein expression was also associated with poor prognosis in prostatectomy-treated patients. Finally, the knock-down of FEN1 with small interfering RNA inhibited the growth of LNCaP cells. Our data demonstrate that the overexpression of AR sensitizes the receptor binding to chromatin, thus, explaining how AR signaling pathway is reactivated in CRPC cells.

Impact of PET/CT on planning of radiotherapy in head and neck cancer.

Radiation therapy (RT) has a prominent role in the treatment of locally advanced head and neck cancer. Image and biologically guided intensity modulated RT are becoming strongholds of state-of-the-art management with positron emission tomography (PET)/computed tomography (CT) as the preferred diagnostic tool in treatment planning. The procedures required in the workflow from diagnosis to treatment plan are complex and consensus on optimal image acquisition, reconstruction parameters and contouring methods remains to be established. In spite of this the potential of PET/CT-based treatment planning has been widely recognized and many large referral centres have adopted the technique in either a routine or an experimental setting. PET/CT with 2-deoxy-2-[¹8F]fluoro-D-glucose ([¹8F]FDG) assists in selection of correct treatment goal and dose optimisation and increases the confidence of contouring process modifying treatment plan in most patients. For dose escalation and adaptive RT strategies PET may provide regional distribution of desired tumour characteristics such as hypoxic, metabolically active or rapidly proliferating sub-volumes. It is expected that within a few years PET/CT will be recommended for all patients presenting with stage III-IV disease considering the obvious benefits associated with more accurate volumetric presentation of primary and locoregional disease and the improved opportunities to conform and escalate RT dose in an attempt to improve therapeutic gain.

Biocompatible photocrosslinked poly(ester anhydride) based on functionalized poly(epsilon-caprolactone) prepolymer shows surface erosion controlled drug release in vitro and in vivo.

Star-shaped poly(epsilon-caprolactone) oligomers functionalized with succinic anhydride were used as prepolymers to prepare photocrosslinked poly(ester anhydride) to evaluate their in vivo drug delivery functionality and biocompatibility. Thus, in this work, erosion, drug release and safety of the photocrosslinked poly(ester anhydride) were examined in vitro and in vivo. A small water-soluble drug, propranolol HCl (M(w) 296 g/mol, solubility 50 mg/ml), was used as the model drug in an evaluation of the erosion controlled release. Drug-free and drug-loaded (10-60% w/w) poly(ester anhydride) discoids eroded in vitro (pH 7.4 buffer, +37 degrees C) linearly within 24-48 h. A strong correlation between the polymer erosion and the linear drug release in vitro was observed, indicating that the release had been controlled by the erosion of the polymer. Similarly, in vivo studies (s.c. implantation of discoids in rats) indicated that surface erosion controlled drug release from the discoids (drug loading 40% w/w). Oligomers did not decrease cell viability in vitro and the implanted discoids (s.c., rats) did not evoke any cytokine activity in vivo. In summary, surface erosion controlled drug release and the safety of photocrosslinked poly(ester anhydride) were demonstrated in this study.

Electrospun multifunctional diclofenac sodium releasing nanoscaffold.

Electrospinning is a method utilized to produce nano-scale fibers for tissue engineering applications. A variety of cells are attracted by nano scale surfaces and structures probably due to the similarity of their natural environment scale. In this study, diclofenac sodium (DS) releasing nanofibers were manufactured via electrospinning process. Poly(95 epsilon-capro/5 D,L-lactide) was dissolved into acetic acid to form a 20% w/v solution. 2% w/w of DS was then added into the polymer solution and stirred homogenously. About 1 g of polymer/drug solution was spun onto the collector under electrostatic conditions. The distance between needle tip and sample collector was arranged to 10 cm and applied electric field was 2 kV/cm. Release rate of DS was measured by using UV/VIS spectrophotometer. Resulted highly porous nanofiber scaffold was about 2 mm thick and the diameter of nanofibers was approximately 130 nm. Structure included in also spheres with approximately diameter of 3.30 microm. About 45% of DS was released during the first 24 hours and after that the release decreased to almost zero value. After 35 days release rate increased. This study revealed that manufacturing of highly porous DS releasing nanoscaffold by electrospinning process is feasible. Having fast DS release rate nanofibrous scaffold made of poly(95 epsilon-capro/5 D,L-lactide) can be of benefit for applications where immediate control of tissue reaction is needed.

Crosslinked poly(epsilon-caprolactone/D,L-lactide)/bioactive glass composite scaffolds for bone tissue engineering.

A series of elastic polymer and composite scaffolds for bone tissue engineering applications were designed. Two crosslinked copolymer matrices with 90/10 and 30/70 mol % of epsilon-caprolactone (CL) and D,L-lactide (DLLA) were prepared with porosities from 45 to 85 vol % and their mechanical and degradation properties were tested. Corresponding composite scaffolds with 20-50 wt % of particulate bioactive glass (BAG) were also characterized. Compressive modulus of polymer scaffolds ranged from 190+/-10 to 900+/-90 kPa. Lactide rich scaffolds absorbed up to 290 wt % of water in 4 weeks and mainly lost their mechanical properties. Caprolactone rich scaffolds absorbed no more than 110 wt % of water in 12 weeks and kept their mechanical integrity. Polymer and composite scaffolds prepared with P(CL/DLLA 90/10) matrix and 60 vol % porosity were further analyzed in simulated body fluid and in osteoblast culture. Cell growth was compromised inside the 2 mm thick three-dimensional scaffold specimens as a static culture model was used. However, composite scaffolds with BAG showed increased osteoblast adhesion and mineralization when compared to neat polymer scaffolds.

In vitro Ca-P precipitation on biodegradable thermoplastic composite of poly(epsilon-caprolactone-co-DL-lactide) and bioactive glass (S53P4).

Bioactive properties of composites containing poly(epsilon-caprolactone-co-DL-lactide) with molar ratio 96/4 and bioactive glass (BAG), S53P4, were tested in vitro. The glass content in the tested materials was 40, 60 or 70 wt%, and two granule size ranges (<45 and 90-315 microm) were used. The composites were analysed for their apatite-forming ability. This was determined as a function of time by the dissolution pattern of Si and Ca ions and structural changes on the specimen surfaces. Composite specimens were immersed in simulated body fluid at 37 degrees C for up to 6 months. The changes in Si and Ca concentrations of the immersion medium were determined with UV-Vis and atomic absorption spectrophotometry. The calcium phosphate precipitation and apatite formation were evaluated by scanning electron microscopy (SEM) and infra-red spectroscopy (IR) using the attenuated total reflectance (ATR) system. The SEM and SEM-EDX analysis of the depositions formed on the composite surfaces was in line with the changes in ion concentrations. The clearest results with IR were seen in the material containing 60 wt% small glass particles. The results indicate that composites containing over 40 wt% BAG granules are bioactive, and that a higher BAG surface area/volume ratio favors the apatite formation in vitro.

Biocompatibility of silver nitrate and ofloxacine coated bioabsorbable SR-PLLA rods.

The purpose of this study was to evaluate the biocompatibility of silver nitrate and ofloxacine coatings of bioresorbable self-reinforced poly-L-lactic acid (SR-PLLA) rods. SR-PLLA rods coated with pure poly(caprolactone-co-L-lactide) or blended with silver nitrate (10, 5 or 2 weight-%) or ofloxacine (5 or 2 weight-%) were implanted in the dorsal muscles of 25 male rabbits. Tissue reactions caused by implantation trauma were seen 1 week after implantation. The positive control and 10 w-% silver nitrate coating showed the most marked reactions 1 month after implantation. Only sparse reactions were seen 6 months after implantation. Tissue reactions were scored semi-quantitatively. As a result of this study, we concluded that silver nitrate or ofloxacine coatings up to five w-% did not alter the good biocompatibility of SR-PLLA essentially. The method may lead to the possibility of preventing bacterial adhesion to urological stents during insertion.

Inability of transforming growth factor-beta 1, combined with a bioabsorbable polymer paste, to promote healing of bone defects in the rat distal femur.

The ability of transforming growth factor-beta 1 (TGF-beta 1) to promote bone formation suggests that it may have potential as a therapeutic agent in bone defects. However, there still exists a need for an effective method of delivering TGF-beta 1 to the site of an osseous defect. In the present study, TGF-beta 1 was embedded in a bioabsorbable polymer paste (a blend of an L-lactide oligomer and a copolymer of epsilon-caprolactone and DL-lactide). The release of TGF-beta 1 from the polymer paste was examined in vitro with an enzyme-linked immunosorbent assay, which showed sustained release of active TGF-beta 1 over a 7-day period. Further, the polymer paste was used to fill a bone defect in the rat distal femur. The amount of TGF-beta 1 per rat was 50 micrograms, while in a control group we used an identical polymer paste without the growth factor. After a follow-up of 1 week and 3 weeks, the femurs were examined radiographically, histologically, histomorphometrically, microradiographically, and were also used for tetracycline-labeling studies. TGF-beta 1 did not enhance healing of the bone defect. A combination of growth factors would probably be a more potent osteoinductor than TGF-beta 1 alone.

Effect of the molecular weight of poly(epsilon-caprolactone-co-DL-lactide) on toremifene citrate release from copolymer/silica xerogel composites.

The purpose of this study was to develop a biodegradable polymeric carrier system for toremifene citrate based on epsilon-caprolactone/DL-lactide copolymers and silica xerogel. The effect of the molecular weight of poly(epsilon-caprolactone-co-DL-lactide) affecting the release rate of toremifene citrate from copolymer/silica xerogel composites was evaluated by in vitro dissolution study. Lower and higher molecular weight copolymers (LMW 60000 g/mol and HMW 300000 g/mol) were used in the devices. Drug release was compared from the (copolymer/drug) matrix device and the (copolymer/drug impregnated silica xerogel) composite device. Hydrolysis of the copolymer devices was evaluated by water absorption, weight loss and change of molecular weight by size exclusion measurements (SEC). Controlled release of toremifene citrate was obtained from both matrix and composite devices and the release rate was most affected by the initial molecular weight of the copolymer. Throughout the study better results were obtained with LMW devices, since drug release was steady for nearly 1 year and no changes in the release rate were observed. The drug release was diffusion controlled from both LMW matrix and composite devices. Incorporation of toremifene citrate into the silica xerogel was found to enhance the drug release rate. The copolymer matrices degraded by random hydrolytic chain scission and, unexpectedly, HMW P(CL/LA) degraded faster than LMW P(CL/LA). The release of toremifene citrate from HMW devices was not complete before the second stage of polymer degradation began.

Bacterial adherence to silver nitrate coated poly-L-lactic acid urological stents in vitro.

The purpose of this study was to see whether it is possible to prevent bacterial adherence to bioabsorbable self-reinforced L-lactic acid polymer (SR-PLLA) urological stents. The SR-PLLA stents were coated with silver nitrate blended epsilon-caprolactone/L-lactide copolymer. The adherence of five bacterial strains (Pseudomonas aeruginosa, Enterococcus faecalis, Proteus mirabilis and two strains of Escherichia coli) to coated and non-coated SR-PLLA wires were tested. It was found that silver nitrate coating prevented the adherence of bacteria (except E. faecalis) to SR-PLLA stents. The preventive effect correlated with the silver nitrate concentration. It was also found that silver nitrate coating reduced the amount of bacteria in ambient urine. In conclusion, silver nitrate coating may reduce stent-associated bacterial infections by preventing the adherence of bacteria. Further studies are needed to confirm its efficacy and safety in clinical practice.

Bacterial adherence to ofloxacin-blended polylactone-coated self-reinforced L-lactic acid polymer urological stents.

OBJECTIVE: To determine whether ofloxacin coating has any effect on bacterial adherence to bioresorbable self-reinforced L-lactic acid polymer (SR-PLLA) urological stents. MATERIALS AND METHODS: SR-PLLA stents were coated with epsilon-caprolactone/L-lactide copolymer blended with ofloxacin at three different concentrations of ofloxacin (0.5, 2 and 5% w/w). The adherence of five bacterial strains (Pseudomonas aeruginosa, Enterococcus faecalis, Proteus mirabilis and two strains of Escherichia coli) to the coated SR-PLLA stents was analysed. Uncoated stent pieces were used as controls. The effect of ofloxacin coating on bacterial growth in the microenvironment of the stent pieces was also analysed. RESULTS: Ofloxacin coating prevented bacterial adherence to SR-PLLA stent material; this effect correlated significantly with the ofloxacin concentration of the caprolactone coating. Ofloxacin coating reduced the amount of bacteria in the microenvironment of the stent, but because of natural resistance, ofloxacin coating had little effect on E. faecalis. CONCLUSION: Except for E. faecalis, ofloxacin coating may reduce stent-associated infections. However, further studies are needed to confirm its biocompatibility and efficacy in clinical use.

GCD quantitation of opiates as propionyl derivatives in blood.

We describe a method using a gas chromatograph with electron ionization detection (GCD) for the simultaneous determination of morphine, codeine, 6-monoacetylmorphine, ethylmorphine, and dihydrocodeine in blood. The method employs propionic anhydride in the presence of triethylamine to propionylate free hydroxyl groups of the opiates in blood. The quantitation is achieved by using GCD with selected ion monitoring of the two most characteristic ions for each analyte. The quantitation limit was 0.01 mg/L and the linearity was 0.01-10 mg/L for dihydrocodeine, ethylmorphine, and 6-monoacetylmorphine. For the other investigated opiates, the quantitation limit was 0.025 mg/L and linearity was 0.025-10 mg/L. The intraday relative standard deviation (RSD) varied from 7.2 to 10% at the 0.5 mg/L level, and the day-to-day RSDs varied from 7.5 to 11% at the 0.85 mg/L level.

In vitro evaluation of biodegradable epsilon-caprolactone-co-D, L-lactide/silica xerogel composites containing toremifene citrate.

Poly(epsilon-caprolactone-co-D,L-lactide) polymers were blended with toremifene citrate or with toremifene citrate impregnated silica xerogel in order to develop a controlled release formulation. The copolymers were synthesized by bulk polymerization and characterized by nuclear magnetic resonance, size exclusion chromatography and differential scanning calorimetry analyses. The in vitro release of toremifene citrate, an antiestrogenic compound, and silica was carried out in simulated body fluid (pH 7.4) containing 0.5 wt% sodium dodecylsulphate at 34 degrees C. The in vitro release studies indicate that the release flux of toremifene citrate increases with increasing weight fraction of caprolactone in the copolymer. Silica xerogel had a minor enhancing effect on the release rate of toremifene citrate. Copolymers containing larger amounts of D,L-lactide (PLA-CL20 and PLA-CL40 copolymers) were not suitable matrices for the delivery of toremifene citrate in a controlled manner because of the burst effect. The fraction of toremifene citrate released from PLA-CL80 matrix increased with the increasing loading of toremifene citrate. The results of the study indicate that the in vitro release of toremifene citrate can be adjusted by varying the polymer composition and also the initial drug loading.

Histological evaluation of poly(L-lactide/epsilon-caprolactone) membrane implanted subcutaneously in rats.

BACKGROUND: Poly(L-lactide/epsilon-caprolactone) [P(L-LA/epsilon-CL)] 50/50 membranes is an absorbable membrane which has been developed recently for possible use in tissue engineering. AIMS: To look at histological tissue reactions to the membrane and its behaviour upon its in vivo implantation in rats. MATERIAL AND METHODS: P(L-LA/e-CL) 50/50 membranes, 0.4 mm thick, were implanted subcutaneously in the dorsal neck of 20 Wistar rats. The rats were followed-up for 1, 3, 6, 12 and 28 months. After sacrifice, subcutaneous tissues with implants were taken as specimens, inspected for any gross abnormality and histological examination was carried out. RESULTS: No significant macroscopic changes were noticed except for two implants that were grossly rounded. Histologically, the membranes had undergone cracking, fragmentation and progressive degradation. Cracks appeared initially at the periphery of the implant. Large cracks progressed mainly in longitudinal planes. Cracks occurred in the middle of implants and led to membrane bending in some cases. "Neomembrane" formation, comprised of fibrous tissue and the implant, was seen. The foreign-body reaction to the membrane involved macrophages and foreign-body giant cells. In one case an acute type of polymorphonuclear cell reaction was observed and in a second case a predominantly lymphocytic reaction was seen at three months. They were thought to be the results of infection. No other adverse effects were seen. CONCLUSIONS: P(L-LA/e-CL) 50/50 membrane was found to be biocompatible when implanted subcutaneously in rats. It was degraded to a great extent but not completely in 28 months.