ctDNA analysis reveals different molecular patterns upon disease progression in patients treated with osimertinib.

BACKGROUND: Several clinical trials have demonstrated the efficacy and safety of osimertinib in advanced non-small-cell lung cancer (NSCLC). However, there is significant unexplained variability in treatment outcome. METHODS: Observational prospective cohort of 22 pre-treated patients with stage IV NSCLC harboring the epidermal growth factor receptor (EGFR) p.T790M resistance mutation and who were treated with osimertinib. Three hundred and twenty-six serial plasma samples were collected and analyzed by digital PCR (dPCR) and next-generation sequencing (NGS). RESULTS: The median progression-free survival (PFS), since the start of osimertinib, was 8.9 [interquartile range (IQR): 4.6-18.0] months. The median treatment durations of sequential gefitinib + osimertinib, afatinib + osimertinib and erlotinib + osimertinib treatments were 30.1, 24.6 and 21.1 months, respectively. The p.T790M mutation was detected in 19 (86%) pre-treatment blood samples. Undetectable levels of the original EGFR-sensitizing mutation after 3 months of treatment were associated with superior PFS (HR: 0.2, 95% CI: 0.05-0.7). Likewise, re-emergence of the original EGFR mutation, alone or together with the p.T790M mutation was significantly associated with shorter PFS (HR: 8.8, 95% CI: 1.1-70.7 and HR: 5.9, 95% CI: 1.2-27.9, respectively). Blood-based monitoring revealed three molecular patterns upon progression to osimertinib: sensitizing+/T790M+/C797S+, sensitizing+/T790M+/C797S-, and sensitizing+/T790M-/C797S-. Median time to progression in patients showing the triplet pattern (sensitizing+/T790M+/C797S+) was 12.27 months compared with 4.87 months in patients in whom only the original EGFR sensitizing was detected, and 2.17 months in patients showing the duplet pattern (sensitizing+/T790M+). Finally, we found that mutations in exon 545 of the PIK3CA gene were the most frequent alteration detected upon disease progression in patients without acquired EGFR-resistance mutations. CONCLUSIONS: Different molecular patterns identified by plasma genotyping may be of prognostic significance, suggesting that the use of liquid biopsy is a valuable approach for tumor monitoring.

Cisplatin resistance involves a metabolic reprogramming through ROS and PGC-1α in NSCLC which can be overcome by OXPHOS inhibition.

BACKGROUND: Platinum-based chemotherapy remains the standard of care for most lung cancer cases. However chemoresistance is often developed during the treatment, limiting clinical utility of this drug. Recently, the ability of tumor cells to adapt their metabolism has been associated to resistance to therapies. In this study, we first described the metabolic reprogramming of Non-Small Cell Lung Cancer (NSCLC) in response to cisplatin treatment. METHODS: Cisplatin-resistant versions of the A549, H1299, and H460 cell lines were generated by continuous drug exposure. The long-term metabolic changes, as well as, the early response to cisplatin treatment were analyzed in both, parental and cisplatin-resistant cell lines. In addition, four Patient-derived xenograft models treated with cisplatin along with paired pre- and post-treatment biopsies from patients were studied. Furthermore, metabolic targeting of these changes in cell lines was performed downregulating PGC-1α expression through siRNA or using OXPHOS inhibitors (metformin and rotenone). RESULTS: Two out of three cisplatin-resistant cell lines showed a stable increase in mitochondrial function, PGC1-α and mitochondrial mass with reduced glycolisis, that did not affect the cell cycle. This phenomenon was confirmed in vivo. Post-treatment NSCLC tumors showed an increase in mitochondrial mass, PGC-1α, and a decrease in the GAPDH/MT-CO1 ratio. In addition, we demonstrated how a ROS-mediated metabolism reprogramming, involving PGC-1α and increased mitochondrial mass, is induced during short-time cisplatin exposure. Moreover, we tested how cells with increased PGC-1a induced by ZLN005 treatment, showed reduced cisplatin-driven apoptosis. Remarkably, the long-term metabolic changes, as well as the metabolic reprogramming during short-time cisplatin exposure can be exploited as an Achilles' heel of NSCLC cells, as demonstrated by the increased sensitivity to PGC-1α interference or OXPHOS inhibition using metformin or rotenone. CONCLUSION: These results describe a new cisplatin resistance mechanism in NSCLC based on a metabolic reprogramming that is therapeutically exploitable through PGC-1α downregulation or OXPHOS inhibitors.

Cancer-associated fibroblasts modify lung cancer metabolism involving ROS and TGF-ß signaling.

Lung cancer is a major public health problem due to its high incidence and mortality rate. The altered metabolism in lung cancer is key for the diagnosis and has implications on both, the prognosis and the response to treatments. Although Cancer-associated fibroblasts (CAFs) are one of the major components of the tumor microenvironment, little is known about their role in lung cancer metabolism. We studied tumor biopsies from a cohort of 12 stage IIIA lung adenocarcinoma patients and saw a positive correlation between the grade of fibrosis and the glycolysis phenotype (Low PGC-1α and High GAPDH/MT-CO1 ratio mRNA levels). These results were confirmed and extended to other metabolism-related genes through the in silico data analysis from 73 stage IIIA lung adenocarcinoma patients available in TCGA. Interestingly, these relationships are not observed with the CAFs marker α-SMA in both cohorts. To characterize the mechanism, in vitro co-culture studies were carried out using two NSCLC cell lines (A549 and H1299 cells) and two different fibroblast cell lines. Our results confirm that a metabolic reprogramming involving ROS and TGF-ß signaling occurs in lung cancer cells and fibroblasts independently of α-SMA induction. Under co-culture conditions, Cancer-Associated fibroblasts increase their glycolytic ability. On the other hand, tumor cells increase their mitochondrial function. Moreover, the differential capability among tumor cells to induce this metabolic shift and also the role of the basal fibroblasts Oxphos Phosphorylation (OXPHOS) function modifying this phenomenon could have implications on both, the diagnosis and prognosis of patients. Further knowledge in the mechanism involved may allow the development of new therapies.

Keeping Things in Balance: Family Experiences of Living With Alzheimer's Disease.

Background and Objectives: Historically, research, practice, and policy on Alzheimer's family care has been largely focused on dyadic relationships and the experience of burden and stress, often failing to capture the complex caring dynamics as they unfold over time. This study sought to develop a more nuanced understanding of how people with Alzheimer's disease (AD) and their families are affected by the disease and how they respond to the challenges AD poses. Research Design and Methods: Using constructivist grounded theory, the study explored the experiences of living with AD for native Spanish families residing in Navarra. Data were collected from a series of 26 interviews with seven family units living with early stage AD and further validated with three focus groups with 14 caregivers of people with mid/advanced AD. Data were analyzed to capture the main processes involved and charted how these evolved over time. Results: Keeping things in balance describes the way family works to preserve a balance between caring and their lives before the onset of AD. Three main temporal stages to the experience of early AD, along with their associated processes, were identified: (a) what's going on here and the processes of noticing, suspecting, and confirming; (b) our life is changing underpinned by the processes of watching and redefining; and (c) keeping things together and its associated balancing process. These reinforced the dynamic nature of living with AD over time. Discussion and Implications: This study was novel in its design as it included the person with AD as part of the functioning family unit. The temporal stages and the key social processes identified have the potential to inform the development of "stage-specific" interventions for the support of the whole family at various points in time.

Life history parameters and scale-cover surface area of Aonidiella aurantii are altered in a mating disruption environment: implications for biological control.

BACKGROUND: In recent years, environmentally safe measures to control the California red scale (CRS), Aonidiella aurantii (Maskell), have been successfully implemented. These measures include mating disruption (MD) and biological control. The goal of this study was to examine the effect of high concentrations of the CRS sex pheromone on its life history parameters and scale-cover surface area under controlled laboratory conditions. RESULTS: The developmental time of both males and females of CRS increased with exposure to airborne pheromone. MD had an effect on both the total number of progeny and on the crawler production period for females. Accordingly, demographic parameters such as net fecundity (R(0) ) and intrinsic rate of increase (r(m) ) were significantly lower in the pheromone-treated populations. The largest scale-cover surface areas were observed in the CRS reared in the pheromone environment. CONCLUSION: A clear influence of airborne pheromone on the biology of CRS has been demonstrated. In addition to the classical mating disruption benefits of this technique, additional benefits, such as increase in the duration of exposure to natural enemies and increase in size, which benefits some species of parasitoids, have been confirmed.

Mating disruption for the control of Aonidiella aurantii Maskell (Hemiptera: Diaspididae) may contribute to increased effectiveness of natural enemies.

BACKGROUND: New directives on sustainable use of pesticides have encouraged research on efficient alternative pest control methods. In the case of the California red scale (CRS), Aonidiella aurantii (Maskell), this imperative, along with the many difficulties in controlling this pest, have led to the investigation of new approaches. Previously developed mating disruption (MD) dispensers, together with the augmentative releases of the parasitoid Aphytis melinus DeBach, are here considered as a combined strategy for use against A. aurantii. RESULTS: Efficacy of MD was demonstrated by a mean reduction of 80% in CRS male catches and a mean fruit damage reduction of 83% compared with the control. A delay in the development of A. aurantii instars was observed in the MD plot. This delay increased the period of exposure of the susceptible instars to natural enemies, which resulted in higher predation and parasitism levels in the MD plot. Under laboratory conditions, A. melinus mating behaviour and effects on A. aurantii were not significantly altered in a CRS-pheromone-saturated environment. CONCLUSION: Mating disruption pheromone did not affect the behaviour or level of parasitism by A. melinus or the incidence of other generalist predators. Therefore, A. aurantii pheromone appears to be compatible with augmentative releases and biological control, making its use a good strategy for CRS management.

Studies on the development of a mating disruption system to control the tomato leafminer, Tuta absoluta Povolny (Lepidoptera: Gelechiidae).

BACKGROUND: The tomato leafminer (Tuta absoluta Povolny) has rapidly colonised the whole Mediterranean and South-Atlantic coasts of Spain, and it has become a key problem in both outdoor and greenhouse crops. New control methods compatible with biological control are required, and mating disruption appears to be a perfect method in current agriculture, as it is an environmentally friendly and residue-free technique. IPM packages tested have included the use of pheromones to detect populations, but there has not been much previous research on mating disruption of T. absoluta. In this work, pheromone doses varying from 10 to 40 g ha(-1), emitted at a constant rate over 4 months, were tested in greenhouses with different levels of containment in order to evaluate the efficacy of mating disruption on T. absoluta. RESULTS: Trials on containment level revealed that the flight of T. absoluta was satisfactorily disrupted with an initial pheromone dose of 30 g ha(-1), and levels of damage did not significantly differ from those in reference plots with insecticide treatments. Later efficacy trials confirmed previous experiences, and release studies showed that control of damage and flight disruption were taking place when releasing at least 85 mg pheromone per ha per day. CONCLUSION: Effective control using pheromone application against T. absoluta can be achieved, in greenhouses with high containment levels, for 4 months, with initial doses of 30 g ha(-1). Further research must be conducted in order to evaluate the prospect of outdoor application of mating disruption systems.

Solid phase microextraction of volatile emissions of Ceratitis capitata (Wiedemann) (Diptera: Tephritidae): influence of fly sex, age, and mating status.

Considerable efforts have been devoted to understanding the courtship behavior and pheromone communication of medflies; however, the sex pheromone composition is still a controversial subject. The discovery of new components affecting medfly behavior would be of interest for medfly control methods based on semiochemicals. This work describes volatile compounds emitted by Ceratitis capitata collected using solid phase microextraction. The volatile study was conducted according to an experimental design with three factors (sex, age, and mating status) assumed to be relevant for better understanding the chemical communication. Emission data were treated by means of principal component analysis, a statistical methodology not previously applied to the study of volatiles emitted by fruit flies. The characterization of emission patterns could be useful for the selection of compounds to be further investigated in biological assays to improve knowledge of the key semiochemicals involved in medfly behavior.

Mating disruption of California red scale, Aonidiella aurantii Maskell (Homoptera: Diaspididae), using biodegradable mesoporous pheromone dispensers.

BACKGROUND: The control of California red scale, Aonidiella aurantii (Maskell), has encountered many difficulties, which has raised interest in alternative control methods. Up to now, the A. aurantii sex pheromone has been used only for monitoring. In a previous work the authors described a biodegradable mesoporous pheromone dispenser for mating disruption. To verify the efficacy of these dispensers, three field trials were conducted, and the results are shown in this paper. RESULTS: The study of the release profile of these dispensers revealed a mean pheromone emission value of 269 microg day(-1) and levels of residual pheromone of 10% at the end of 250 days. During the second flight, an A. aurantii male catch reduction of 98% was achieved in the mating disruption plot of trial 1, 93.5% in trial 2 and 76.7% in trial 3. During the third flight, reductions were 94.1, 82.9 and 68.1% in trials 1, 2 and 3 respectively. Considering damaged fruit with more than five scales, reductions of about 80 and 60% were obtained in the mating disruption plots of trials 2 and 3, respectively, compared with an untreated plot, and a reduction of about 70% in trial 1 compared with an oil-treated plot. CONCLUSION: Mating disruption has been found to be an efficient technique to control this pest, working equally well to a correctly sprayed oil treatment. Further studies are needed to improve the determination of the time of dispenser application and evaluate the effects of the pheromone on natural enemies.

Mediterranean fruit fly suppression using chemosterilants for area-wide integrated pest management.

BACKGROUND: The chemosterilisation technique has been demonstrated to reduce the population and fruit damage of the Mediterranean fruit fly, Ceratitis capitata (Wiedemann), in citrus orchards. Field trials showed efficacy by reducing the fruit fly population, which was progressively achieved by continuous application of lufenuron to several generations. Different authors have suggested that field trials should be carried out in isolated or wide areas in order to reduce fruit fly intrusion and obtain best results. To this end, a wide-area trial over 3600 hectares has been under investigation in Valencia (Spain) since 2002 to validate the chemosterilisation technique against the fruit fly. The whole area was treated with 24 traps ha(-1), using more than 86,000 traps in the field trial. RESULTS: A continuous decrease in fruit fly population was observed over the 4 years under trial. Moreover, results showed a significant reduction in persimmon damage in the chemosterilant treatment area compared with a malathion aerial treatment area. In the case of citrus damage, no significant differences were obtained between malathion and chemosterilant treatments. CONCLUSION: The chemosterilant method reduces Mediterranean fruit fly populations, and therefore it is a candidate treatment to replace aerial treatments with insecticides in order to suppress this pest. In addition, the efficacy of chemosterilant treatment is increasing year after year. The possibility of using this technique combined with other control methods is discussed.

Study on the optimum pheromone release rate for attraction of Chilo suppressalis (Lepidoptera: Pyralidae).

Traditional chemical control against Chilo suppressalis Walker is currently being replaced in Spain by new methods based on pheromones. A key step to improve the efficacy of these methods is the determination of the optimum pheromone release rate, which is still uncertain for this pest. In this work, the pheromone release profile and the field performance of a new mesoporous dispenser was compared with a standard commercial dispenser. For this purpose, pheromone loads were extracted from field-aged dispensers and quantified by gas chromatography with flame ionization detector. In addition, a field trial was carried out with traps baited with one, two, or three mesoporous dispensers per trap, as well as with traps containing one standard dispenser. We found that the highest number of field catches did not correspond to the highest pheromone emission rate, which suggests a repellent effect of the insect if the emission is excessive. The results suggest that the attractant activity was maximized by emitting approximately 34 microg/d. The efficacy of the mesoporous dispenser and its possible improvements are discussed.

Screening for metabolites from Penicillium novae-zeelandiae displaying radical-scavenging activity and oxidative mutagenicity: isolation of gentisyl alcohol.

In the search for new natural products with anti-oxidant activity, we have combined the cell-free assay based on the scavenging of the stable radical 2,2-diphenyl-1-picrylhydrazyl (DPPH), with a bioassay that detects oxidative mutagens. This bioassay uses a new Escherichia coli tester strain, IC203, specifically sensitive to oxidative stress due to a deficiency in the OxyR function. OxyR is a redox-sensitive transcriptional activator of genes encoding anti-oxidant enzymes such as catalase and peroxiredoxin alkyl hydroperoxide reductase. The positive response observed in E. coli IC203 with several known anti-oxidants, including cysteine, catechol and ascorbic acid, suggested to us the usefulness of the mutagenicity assay for a rapid screening of anti-oxidant compounds. The extract from Penicillium novae-zeelandiae was found to scavenge the DPPH radical. Subsequently, guided by the DPPH-scavenging assay and the oxidative mutagenesis assay, we isolated and identified three compounds in fractions from that active extract: patulin (1). 3-hydroxybenzyl alcohol (2). and gentisyl alcohol (2,5-dihydroxybenzyl alcohol) (3). Of these, gentisyl alcohol showed both DPPH-scavenging activity and oxidative mutagenicity. This compound also gave rise to intracellular formation of superoxide, evaluated by monitoring the oxidation of dihydroethidium, and was able to inhibit mutagenesis induced by the model oxidant t-butyl hydroperoxide (t-BuOOH).