Metallothionein-3 is a multifunctional driver that modulates the development of sorafenib-resistant phenotype in hepatocellular carcinoma cells.

BACKGROUND & AIMS: Metallothionein-3 (hMT3) is a structurally unique member of the metallothioneins family of low-mass cysteine-rich proteins. hMT3 has poorly characterized functions, and its importance for hepatocellular carcinoma (HCC) cells has not yet been elucidated. Therefore, we investigated the molecular mechanisms driven by hMT3 with a special emphasis on susceptibility to sorafenib. METHODS: Intrinsically sorafenib-resistant (BCLC-3) and sensitive (Huh7) cells with or without up-regulated hMT3 were examined using cDNA microarray and methods aimed at mitochondrial flux, oxidative status, cell death, and cell cycle. In addition, in ovo/ex ovo chick chorioallantoic membrane (CAM) assays were conducted to determine a role of hMT3 in resistance to sorafenib and associated cancer hallmarks, such as angiogenesis and metastastic spread. Molecular aspects of hMT3-mediated induction of sorafenib-resistant phenotype were delineated using mass-spectrometry-based proteomics. RESULTS: The phenotype of sensitive HCC cells can be remodeled into sorafenib-resistant one via up-regulation of hMT3. hMT3 has a profound effect on mitochondrial respiration, glycolysis, and redox homeostasis. Proteomic analyses revealed a number of hMT3-affected biological pathways, including exocytosis, glycolysis, apoptosis, angiogenesis, and cellular stress, which drive resistance to sorafenib. CONCLUSIONS: hMT3 acts as a multifunctional driver capable of inducing sorafenib-resistant phenotype of HCC cells. Our data suggest that hMT3 and related pathways could serve as possible druggable targets to improve therapeutic outcomes in patients with sorafenib-resistant HCC.

Discovery, Optimization, and Biological Evaluation of Arylpyridones as Cbl-b Inhibitors.

Casitas B-lymphoma proto-oncogene-b (Cbl-b), a member of the Cbl family of RING finger E3 ubiquitin ligases, has been demonstrated to play a central role in regulating effector T-cell function. Multiple studies using gene-targeting approaches have provided direct evidence that Cbl-b negatively regulates T, B, and NK cell activation via a ubiquitin-mediated protein modulation. Thus, inhibition of Cbl-b ligase activity can lead to immune activation and has therapeutic potential in immuno-oncology. Herein, we describe the discovery and optimization of an arylpyridone series as Cbl-b inhibitors by structure-based drug discovery to afford compound 31. This compound binds to Cbl-b with an IC50 value of 30 nM and induces IL-2 production in T-cells with an EC50 value of 230 nM. Compound 31 also shows robust intracellular target engagement demonstrated through inhibition of Cbl-b autoubiquitination, inhibition of ubiquitin transfer to ZAP70, and the cellular modulation of phosphorylation of a downstream signal within the TCR axis.

Discovery of a Novel Benzodiazepine Series of Cbl-b Inhibitors for the Enhancement of Antitumor Immunity.

Casitas B-lineage lymphoma proto-oncogene-b (Cbl-b) is a RING finger E3 ligase that is responsible for repressing T-cell, natural killer (NK) cell, and B-cell activation. The robust antitumor activity observed in Cbl-b deficient mice arising from elevated T-cell and NK-cell activity justified our discovery effort toward Cbl-b inhibitors that might show therapeutic promise in immuno-oncology, where activation of the immune system can drive the recognition and killing of cancer cells. We undertook a high-throughput screening campaign followed by structure-enabled optimization to develop a novel benzodiazepine series of potent Cbl-b inhibitors. This series displayed nanomolar levels of biochemical potency, as well as potent T-cell activation. The functional activity of this class of Cbl-b inhibitors was further corroborated with ubiquitin-based cellular assays.

Segmenting computed tomograms for cardiac ablation using machine learning leveraged by domain knowledge encoding.

Background: Segmentation of computed tomography (CT) is important for many clinical procedures including personalized cardiac ablation for the management of cardiac arrhythmias. While segmentation can be automated by machine learning (ML), it is limited by the need for large, labeled training data that may be difficult to obtain. We set out to combine ML of cardiac CT with domain knowledge, which reduces the need for large training datasets by encoding cardiac geometry, which we then tested in independent datasets and in a prospective study of atrial fibrillation (AF) ablation. Methods: We mathematically represented atrial anatomy with simple geometric shapes and derived a model to parse cardiac structures in a small set of N = 6 digital hearts. The model, termed "virtual dissection," was used to train ML to segment cardiac CT in N = 20 patients, then tested in independent datasets and in a prospective study. Results: In independent test cohorts (N = 160) from 2 Institutions with different CT scanners, atrial structures were accurately segmented with Dice scores of 96.7% in internal (IQR: 95.3%-97.7%) and 93.5% in external (IQR: 91.9%-94.7%) test data, with good agreement with experts (r = 0.99; p < 0.0001). In a prospective study of 42 patients at ablation, this approach reduced segmentation time by 85% (2.3 ± 0.8 vs. 15.0 ± 6.9 min, p < 0.0001), yet provided similar Dice scores to experts (93.9% (IQR: 93.0%-94.6%) vs. 94.4% (IQR: 92.8%-95.7%), p = NS). Conclusions: Encoding cardiac geometry using mathematical models greatly accelerated training of ML to segment CT, reducing the need for large training sets while retaining accuracy in independent test data. Combining ML with domain knowledge may have broad applications.

Coping with Transgressions by the Romantic Partner: An Inventory Validation and its Correlation with Forgiveness / Afrontamiento ante las Transgresiones de la Pareja: Validación de un Inventario y su Relación con el Perdón

Abstract Almost every person has to deal with transgressions committed by a romantic partner and faces their negative psychological outcomes, and coping strategies might be key to understanding post-transgression dynamics and forgiveness. We tested the construct validity of the Inventory of Strategies for Coping with a Partner's Transgression which include the emotion (E-FCS), problem (P-FCS), and meaning-focused coping strategies (M-FCS) scales. Results replicated the factor structure of each of the three scales through confirmatory factor analysis techniques, tested its reliability with the McDonald's omega coefficient, and then correlated the scales with forgiveness and resentment, strengthening its construct validity. In general, the validity and reliability of scales were confirmed. Emotion-focused strategies showed a negative correlation with forgiveness, while problem and meaning-focused strategies had a positive correlation. These findings were discussed in the context of theory and their practical implications.

Resumen Las personas tienden a lidiar con transgresiones cometidas por su pareja y enfrentar sus consecuencias psicológicas aversivas, por lo que las estrategias de afrontamiento pueden ser clave para la dinámica post-transgresión, y el perdón. Validamos tres escalas para medir estrategias de afrontamiento ante las transgresiones cometidas por la pareja: Estrategias enfocadas en la emoción (E-FCS), el problema (P-FCS) y el sentido (M-FCS). Los resultados muestran que las estructuras factoriales se replicaron en mediante análisis factoriales confirmatorios, se puso a prueba su confiabilidad mediante el coeficiente omega de McDonald, y finalmente se correlacionaron con el perdón y resentimiento, fortaleciendo su validez de constructo. En general los resultados muestran evidencia de validez de constructo y confiabilidad, en general los factores de la E-FCS se correlacionaron negativamente con al perdón, mientras que de P-FCS Y M-FSC lo hicieron positivamente. Se discuten los hallazgos a la luz de la teoría, y sus implicaciones prácticas.

Survival benefit of pediatric-based regimen for adults with acute lymphoblastic leukemia: A single-center retrospective cohort

ABSTRACT Introduction: Improving survival of Acute Lymphoblastic Leukemia (ALL) in adult patients has been a challenge. Despite intensive chemotherapy treatment, overall survival is poor. However, several studies demonstrate that young adult patients have better survival when treated with pediatric-based intensive regimens. Considering these results, We decided to treat newly diagnosed ALL patients according to age and risk factors. The goal of this study was to describe the results of this intensive chemotherapy treatment approach for ALL adult patients diagnosed at our institution. Methods: Fifty-eight ALL patients, diagnosed from 2004 to 2013, were included in the analysis. Patients were assigned to either the St. Jude Total Therapy XIIIB high-risk arm (St Jude) or the CALGB 8811 (CALGB). The Kaplan-Meier survival curve was used for the survival analyses and the Cox proportional hazard regression, for multivariable analysis. Results: The overall survival was 22.9% at 10 years. The St. Jude improved survival, compared to the CALGB (p = 0.007), with 32.6% vs. 7.4% survival rate at 10 years. However, no survival benefit was found for patients younger than 20 years old (p = 0.32). The multivariable analysis demonstrated that undetectable minimal residual disease (MRD) and hematopoietic stem cell transplantation (HSCT) had beneficial impact on survival (p = 0.0007 and p = 0.004, respectively). Conclusion: ALL is a disease of poor prognosis for adults. The joint effort to standardize treatment and seek solutions is the way to start improving this scenario.

The early impact of diets enriched with saturated and unsaturated fatty acids on intestinal inflammation and tight junctions.

The gut has been suggested as the first organ to be affected by unbalanced diets contributing to the obesogenic process. This study aimed to test a short time-course exposition model to a known pro- or anti-inflammatory enriched fatty diet to understand the early gut alterations. Male mice were exposed to the chow diet (CT), high-fat (HF) diet, or a high-fat diet partially replaced on flaxseed oil (FS), rich in omega-3 (ω3), for 14 days. HF and FS increased the total body weight mass compared with the CT group, but FS reduced the epididymal fat depot compared to HF. The bioinformatics from mice and human databases showed the Zo1-Ocln-Cldn7 tight junctions as the main protein-triad. In the ileum, the HF diet has increased IL1ß transcript and IL1ß, TNFα, and CD11b proteins, but reduced the tight junctions (Zo1, Ocln, and Cld7) compared to the CT group. Despite the FS diet being partially efficient in protecting the ileum against inflammation, the tight junctions were increased, compared to the HF group. The GPR120 and GPR40 receptors were unaffected by diets, but GPR120 was colocalized on the surface of ileum macrophages. The short period of a high-fat diet was enough to start the obesogenic process, ileum inflammation, and reduce the tight junctions. Flaxseed oil did not protect efficiently against dysmetabolism. Still, it increased the tight junctions, even without alteration on inflammatory parameters, suggesting the protection against gut permeability during early obesity development.

Quantifying a spectrum of clinical response in atrial tachyarrhythmias using spatiotemporal synchronization of electrograms.

AIMS: There is a clinical spectrum for atrial tachyarrhythmias wherein most patients with atrial tachycardia (AT) and some with atrial fibrillation (AF) respond to ablation, while others do not. It is undefined if this clinical spectrum has pathophysiological signatures. This study aims to test the hypothesis that the size of spatial regions showing repetitive synchronized electrogram (EGM) shapes over time reveals a spectrum from AT, to AF patients who respond acutely to ablation, to AF patients without acute response. METHODS AND RESULTS: We studied n = 160 patients (35% women, 65.0 ± 10.4 years) of whom (i) n = 75 had AF terminated by ablation propensity matched to (ii) n = 75 without AF termination and (iii) n = 10 with AT. All patients had mapping by 64-pole baskets to identify areas of repetitive activity (REACT) to correlate unipolar EGMs in shape over time. Synchronized regions (REACT) were largest in AT, smaller in AF termination, and smallest in non-termination cohorts (0.63 ± 0.15, 0.37 ± 0.22, and 0.22 ± 0.18, P < 0.001). Area under the curve for predicting AF termination in hold-out cohorts was 0.72 ± 0.03. Simulations showed that lower REACT represented greater variability in clinical EGM timing and shape. Unsupervised machine learning of REACT and extensive (50) clinical variables yielded four clusters of increasing risk for AF termination (P < 0.01, χ2), which were more predictive than clinical profiles alone (P < 0.001). CONCLUSION: The area of synchronized EGMs within the atrium reveals a spectrum of clinical response in atrial tachyarrhythmias. These fundamental EGM properties, which do not reflect any predetermined mechanism or mapping technology, predict outcome and offer a platform to compare mapping tools and mechanisms between AF patient groups.

Chick chorioallantoic membrane (CAM) assay for the evaluation of the antitumor and antimetastatic activity of platinum-based drugs in association with the impact on the amino acid metabolism.

The combination of in ovo and ex ovo chorioallantoic membrane (CAM) assay provides an excellent platform which extends its relevance in studying carcinogenesis to the field of screening of anticancer activity of platinum nanoparticles (PtNPs) and further study of the amino acids' fluctuations in liver and brain. PtNPs are promising candidates for replacing cisplatin (CDDP); however, insufficient data of their antitumor efficiency and activity on the cancer-related amino acid metabolism are available, and the assessment of the in vivo performance has barely scratched the surface. Herein, we used CAM assay as in vivo model for screening of novel therapeutic modalities, and we conducted a comparative study of the effects of CDDP and polyvinylpyrrolidone coated PtNPs on MDA-MB-231 breast cancer xenograft. PtNPs showed a higher efficiency to inhibit the tumor growth and metastasis compared to CDDP. The amino acids profiling in the MDA-MB-231 â€‹cells revealed that the PtNPs had an overall depleting effect on the amino acids content. Noteworthy, more side effects to amino acid metabolism were deduced from the depletion of the amino acids in tumor, brain, and liver upon CDDP treatment. Different sets of enzymes of the tricarboxylic acid (TCA) cycle were targeted by PtNPs and CDDP, and while mRNA encoding multiple enzymes was downregulated by PtNPs, the treatment with CDDP affected only two TCA enzymes, indicating a different mechanism of action. Taken together, CAM assay represents and invaluable model, demonstrating the PtNPs capability of repressing angiogenesis, decrease amino acid contents and disrupt the TCA cycle.

Survival benefit of pediatric-based regimen for adults with acute lymphoblastic leukemia: A single-center retrospective cohort.

INTRODUCTION: Improving survival of Acute Lymphoblastic Leukemia (ALL) in adult patients has been a challenge. Despite intensive chemotherapy treatment, overall survival is poor. However, several studies demonstrate that young adult patients have better survival when treated with pediatric-based intensive regimens. Considering these results, We decided to treat newly diagnosed ALL patients according to age and risk factors. The goal of this study was to describe the results of this intensive chemotherapy treatment approach for ALL adult patients diagnosed at our institution. METHODS: Fifty-eight ALL patients, diagnosed from 2004 to 2013, were included in the analysis. Patients were assigned to either the St. Jude Total Therapy XIIIB high-risk arm (St Jude) or the CALGB 8811 (CALGB). The Kaplan-Meier survival curve was used for the survival analyses and the Cox proportional hazard regression, for multivariable analysis. RESULTS: The overall survival was 22.9% at 10 years. The St. Jude improved survival, compared to the CALGB (p = 0.007), with 32.6% vs. 7.4% survival rate at 10 years. However, no survival benefit was found for patients younger than 20 years old (p = 0.32). The multivariable analysis demonstrated that undetectable minimal residual disease (MRD) and hematopoietic stem cell transplantation (HSCT) had beneficial impact on survival (p = 0.0007 and p = 0.004, respectively). CONCLUSION: ALL is a disease of poor prognosis for adults. The joint effort to standardize treatment and seek solutions is the way to start improving this scenario.

TDO2-augmented fibroblasts secrete EVs enriched in immunomodulatory Y-derived small RNA.

Mounting evidence implicates extracellular vesicles (EVs) factors as mediators of cell therapy. Cardiosphere-derived cells are cardiac-derived cells with tissue reparative capacity. Activation of a downstream target of wnt/ß-catenin signalling, tryptophan 2,3 dioxygenase (TDO2) renders therapeutically inert skin fibroblasts cardioprotective. Here, we investigate the mechanism by which concentrated conditioned media from TDO2-augmented fibroblasts (TDO2-CCM) exert cardioprotective effects. TDO2-CCM is cardioprotective in a mouse model of MI compared to CCM from regular fibroblasts (HDF-CCM). Transcriptomic analysis of cardiac tissue at 24 h demonstrates broad suppression of inflammatory and cell stress markers in animals given TDO2-CCM compared to HDF-CCM or vehicle. Sequencing analysis of TDO2-EV RNA demonstrated abundance of a small Y-derived small RNA dubbed 'NT4'. Purification of TDO2-EVs by size-exclusion chromatography and RNAse protection assays demonstrated that NT4 is encapsulated inside EVs. Consistently with TDO2-CCM, macrophages exposed to NT4 showed suppression of the inflammatory and cell stress mediators, particularly p21/cdkn1a. NT4-depleted TDO2-CCM resulted in diminished immunomodulatory capacity. Finally, administration of NT4 alone was cardioprotective in an acute model of myocardial infarction. Taken together, these findings elucidate the mechanism by which TDO2 augmentation mediates potency in secreted EVs through enrichment of NT4 which suppresses upstream cell stress mediators including p21/cdkn1a.

Predicting acute termination and non-termination during ablation of human atrial fibrillation using quantitative indices.

Background: Termination of atrial fibrillation (AF), the most common arrhythmia in the United States, during catheter ablation is an attractive procedural endpoint, which has been associated with improved long-term outcome in some studies. It is not clear, however, whether it is possible to predict termination using clinical data. We developed and applied three quantitative indices in global multielectrode recordings of AF prior to ablation: average dominant frequency (ADF), spectral power index (SPI), and electrogram quality index (EQI). Methods: In N = 42 persistent AF patients (65 ± 9 years, 14% female) we collected unipolar electrograms from 64-pole baskets (Abbott, CA). We studied N = 17 patients in whom AF terminated during ablation ("Term") and N = 25 in whom it did not ("Non-term"). For each index, we determined its ability to predict ablation by computing receiver operating characteristic (ROC) and calculated the area under the curve (AUC). Results: The ADF did not differ for Term and Non-term patients at 5.28 ± 0.82 Hz and 5.51 ± 0.81 Hz, respectively (p = 0.34). Conversely, the SPI for these two groups was. 0.85 (0.80-0.92) and 0.97 (0.93-0.98) and the EQI was 0.61 (0.58-0.64) and 0.56 (0.55-0.59) (p < 0.0001). The AUC for predicting AF termination for the SPI was 0.85 ([0.68, 0.95] 95% CI), and for the EQI, 0.86 ([0.72, 0.95] 95% CI). Conclusion: Both the EQI and the SPI may provide a useful clinical tool to predict procedural ablation outcome in persistent AF patients. Future studies are required to identify which physiological features of AF are revealed by these indices and hence linked to AF termination or non-termination.

Identification of atrial fibrillation drivers by means of concentric ring electrodes.

BACKGROUND AND OBJECTIVE: The prevalence of atrial fibrillation (AF) has tripled in the last 50 years due to population aging. High-frequency (DFdriver) activated atrial regions lead the activation of the rest of the atria, disrupting the propagation wavefront. Fourier based spectral analysis of body surface potential maps have been proposed for DFdriver identification, although these approaches present serious drawbacks due to their limited spectral resolution for short AF epochs and the blurring effect of the volume conductor. Laplacian signals (BC-ECG) from bipolar concentric ring electrodes (CRE) have been shown to outperform the spatial resolution achieved with conventional unipolar recordings. Our aimed was to determine the best DFdriver estimator in endocardial electrograms and to assess the BC-ECG capacity of CRE to quantify AF activity non-invasively. METHODS: 31 AF episodes were simulated using realistic tridimensional models of the atria electrical activity and torso. Periodogram and autoregressive (AR) spectral estimators were computed and the percentile (P90th, P95th and P98th) to impose on the dominant frequencies (DFs) across whole atria to define the best DFdriver estimator evaluated. The identification of DFdriver on DFs from BC-ECG and unipolar surface signals with conventional disc electrodes was compared. RESULTS: The best DFdriver estimator was P95th and AR order 100. BC-ECG signals allowed better detection of AF activity than unipolar signals, with a significantly greater percentage of electrode locations in which DFdriver was identified (p-value 0.0095). CONCLUSIONS: The use of BC-ECG signals for body surface Laplacian potential mapping with CRE could be helpful for better AF diagnosis, prognosis and ablation procedures than those with conventional disk electrodes.

Correction: The Zinc-Schiff Base-Novicidin Complex as a Potential Prostate Cancer Therapy.

[This corrects the article DOI: 10.1371/journal.pone.0163983.].

From 12 to 1 ECG lead: multiple cardiac condition detection mixing a hybrid machine learning approach with a one-versus-rest classification strategy.

Objective. Detecting different cardiac diseases using a single or reduced number of leads is still challenging. This work aims to provide and validate an automated method able to classify ECG recordings. Performance using complete 12-lead systems, reduced lead sets, and single-lead ECGs is evaluated and compared.Approach. Seven different databases with 12-lead ECGs were provided during thePhysioNet/Computing in Cardiology Challenge2021, where 88 253 annotated samples associated with none, one, or several cardiac conditions among 26 different classes were released for training, whereas 42 896 hidden samples were used for testing. After signal preprocessing, 81 features per ECG-lead were extracted, mainly based on heart rate variability, QRST patterns and spectral domain. Next, a One-versus-Rest classification approach made of independent binary classifiers for each cardiac condition was trained. This strategy allowed each ECG to be classified as belonging to none, one or several classes. For each class, a classification model among two binary supervised classifiers and one hybrid unsupervised-supervised classification system was selected. Finally, we performed a 3-fold cross-validation to assess the system's performance.Main results. Our classifiers received scores of 0.39, 0.38, 0.39, 0.38, and 0.37 for the 12, 6, 4, 3 and 2-lead versions of the hidden test set with the Challenge evaluation metric (CM). Also, we obtained a meanG-score of 0.80, 0.78, 0.79, 0.79, 0.77 and 0.74 for the 12, 6, 4, 3, 2 and 1-lead subsets with the public training set during our 3-fold cross-validation.Significance. We proposed and tested a machine learning approach focused on flexibility for identifying multiple cardiac conditions using one or more ECG leads. Our minimal-lead approach may be beneficial for novel portable or wearable ECG devices used as screening tools, as it can also detect multiple and concurrent cardiac conditions.

Atrial fibrillation signatures on intracardiac electrograms identified by deep learning.

BACKGROUND: Automatic detection of atrial fibrillation (AF) by cardiac devices is increasingly common yet suboptimally groups AF, flutter or tachycardia (AT) together as 'high rate events'. This may delay or misdirect therapy. OBJECTIVE: We hypothesized that deep learning (DL) can accurately classify AF from AT by revealing electrogram (EGM) signatures. METHODS: We studied 86 patients in whom the diagnosis of AF or AT was established at electrophysiological study (25 female, 65 ± 11 years). Custom DL architectures were trained to identify AF using N = 29,340 unipolar and N = 23,760 bipolar EGM segments. We compared DL to traditional classifiers based on rate or regularity. We explained DL using computer models to assess the impact of controlled variations in shape, rate and timing on AF/AT classification in 246,067 EGMs reconstructed from clinical data. RESULTS: DL identified AF with AUC of 0.97 ± 0.04 (unipolar) and 0.92 ± 0.09 (bipolar). Rule-based classifiers misclassified ∼10-12% of cases. DL classification was explained by regularity in EGM shape (13%) or timing (26%), and rate (60%; p < 0.001), and also by a set of unipolar EGM shapes that classified as AF independent of rate or regularity. Overall, the optimal AF 'fingerprint' comprised these specific EGM shapes, >15% timing variation, <0.48 correlation in beat-to-beat EGM shapes and CL < 190 ms (p < 0.001). CONCLUSIONS: Deep learning of intracardiac EGMs can identify AF or AT via signatures of rate, regularity in timing or shape, and specific EGM shapes. Future work should examine if these signatures differ between different clinical subpopulations with AF.

Water reuse in the post-tanning process: minimizing environmental impact of leather production.

Post-tanning wastewater is very diversified, as the post-tanning stage should meet the desirable properties of the leather for the final product, with low standardization of the process (compared to beamhouse and tanning). This makes post-tanning effluent reuse less feasible, and reuse in the post-tanning stage still needs to be explored. This work aims to evaluate the reuse of liquid effluents in the post-tanning process. The work methodology consisted of (i) characterization of water streams (groundwater, liquid effluent after primary treatment, and liquid effluent after secondary treatment); (ii) pilot-scale post-tanning tests using groundwater, primary effluent, and secondary effluent; (iii) characterization of the residual baths from pilot-scale tests (pH, conductivity, total solids, chemical oxygen demand, biochemical oxygen demand, chloride, hardness and oil and grease); and (iv) testing the leather obtained for total sulfated ash and organoleptic properties. Results showed that the primary effluent and the secondary effluent could be reused in pilot-scale post-tanning tests. There was an increase in the conductivity of the residual baths when liquid effluents were reused, which confirms the accumulation of salts in the effluents after their reuse.

Extending the Applicability of In Ovo and Ex Ovo Chicken Chorioallantoic Membrane Assays to Study Cytostatic Activity in Neuroblastoma Cells.

PURPOSE: The chick chorioallantoic membrane (CAM) assay can provide an alternative versatile, cost-effective, and ethically less controversial in vivo model for reliable screening of drugs. In the presented work, we demonstrate that CAM assay (in ovo and ex ovo) can be simply employed to delineate the effects of cisplatin (CDDP) and ellipticine (Elli) on neuroblastoma (Nbl) cells in terms of their growth and metastatic potential. METHODS: The Nbl UKF-NB-4 cell line was established from recurrent bone marrow metastases of high-risk Nbl (stage IV, MYCN amplification, 7q21 gain). Ex ovo and in ovo CAM assays were optimized to evaluate the antimetastatic activity of CDDP and Elli. Immunohistochemistry, qRT-PCR, and DNA isolation were performed. RESULTS: Ex ovo CAM assay was employed to study whether CDDP and Elli exhibit any inhibitory effects on growth of Nbl xenograft in ex ovo CAM assay. Under the optimal conditions, Elli and CDDP exhibited significant inhibition of the size of the primary tumor. To study the efficiency of CDDP and Elli to inhibit primary Nbl tumor growth, intravasation, and extravasation in the organs, we adapted the in ovo CAM assay protocol. In in ovo CAM assay, both studied compounds (CDDP and Elli) exhibited significant (p < 0.001) inhibitory activity against extravasation to all investigated organs including distal CAM. CONCLUSIONS: Taken together, CAM assay could be a helpful and highly efficient in vivo approach for high-throughput screening of libraries of compounds with expected anticancer activities.

Prognostic Score and Benefit from Abiraterone in First-line Metastatic, Castration-resistant Prostate Cancer.

BACKGROUND: Most available prognostic nomograms in metastatic castration-resistant prostate cancer (mCRPC) are derived from datasets not representative of the current treatment landscape. A prognostic nomogram for first-line mCRPC treatment was developed from patients treated in the PREVAIL study. OBJECTIVE: To validate the Armstrong model in the COU-AA-302 trial. DESIGN, SETTING, AND PARTICIPANTS: A post hoc analysis of mCRPC patients treated in the COU-AA-302 trial was carried out (NCT00887198). OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: The Armstrong prognostic model was applied to patients treated in COU-AA-302. A continuous risk score was derived from coefficients from the original model. Time-dependent area under the curve (tAUC) was used to evaluate the overall predictive ability of the model. Patients were categorized according to the number of risk factors present into those at a low (three or fewer risk factors), intermediate (four to six risk factors), and high (seven to ten risk factors) risk. The association with survival was assessed with Cox regression models. Interaction tests were used to assess the impact of treatment arm in each of the prognostic groups. RESULTS AND LIMITATIONS: A total of 1088 patients were analyzed. The risk score was associated with overall survival (OS; tAUC 0.733). Most patients were at a low (49%) or intermediate (41%) risk. Risk category was significantly associated with OS (hazard ratio [HR]: 2.3; 95% confidence interval [CI]: 1.9-2.4; p < 0.001), radiographic progression-free survival (rPFS; HR: 1.7; 95% CI: 1.5-1.8; p < 0.001), and prostate-specific antigen progression-free survival (HR: 1.7; 95% CI: 1.5-1.9; p < 0.001). A significant interaction between risk group and OS (p = 0.007) and rPFS (p = 0.009) was observed. Survival was superior in low-risk patients (HR: 0.73; 95% CI: 0.59-0.89; p = 0.009), but similar in intermediate-risk (HR: 0.97; 95% CI: 0.79-1.21; p = 0.9) and high-risk (HR: 1.35; 95% CI: 0.80-2.28; p = 0.5) patients. Two-year OS rates in abiraterone versus placebo were 82% versus 74% in low-risk, 55% versus 52% in intermediate-risk, and 28% versus 31% in high-risk patients. CONCLUSIONS: We validate the prognostic value of the Armstrong risk model in patients treated with first-line androgen receptor signaling inhibitors. Abiraterone provided a greater benefit in low-risk patients with less aggressive disease. Further research is needed to establish the role of Armstrong risk groups for treatment selection in mCRPC patients. PATIENT SUMMARY: In this report, we validated the Armstrong nomogram in the COU-AA-302 trial population. We found a similar prognostic performance to that of the original model. Good-risk patients received the greatest benefit from abiraterone.