The clinical landscape of cell-free DNA alterations in 1671 patients with advanced biliary tract cancer.

BACKGROUND: Targeted therapies have transformed clinical management of advanced biliary tract cancer (BTC). Cell-free DNA (cfDNA) analysis is an attractive approach for cancer genomic profiling that overcomes many limitations of traditional tissue-based analysis. We examined cfDNA as a tool to inform clinical management of patients with advanced BTC and generate novel insights into BTC tumor biology. PATIENTS AND METHODS: We analyzed next-generation sequencing data of 2068 cfDNA samples from 1671 patients with advanced BTC generated with Guardant360. We carried out clinical annotation on a multi-institutional subset (n = 225) to assess intra-patient cfDNA-tumor concordance and the association of cfDNA variant allele fraction (VAF) with clinical outcomes. RESULTS: Genetic alterations were detected in cfDNA in 84% of patients, with targetable alterations detected in 44% of patients. Fibroblast growth factor receptor 2 (FGFR2) fusions, isocitrate dehydrogenase 1 (IDH1) mutations, and BRAF V600E were clonal in the majority of cases, affirming these targetable alterations as early driver events in BTC. Concordance between cfDNA and tissue for mutation detection was high for IDH1 mutations (87%) and BRAF V600E (100%), and low for FGFR2 fusions (18%). cfDNA analysis uncovered novel putative mechanisms of resistance to targeted therapies, including mutation of the cysteine residue (FGFR2 C492F) to which covalent FGFR inhibitors bind. High pre-treatment cfDNA VAF was associated with poor prognosis and shorter response to chemotherapy and targeted therapy. Finally, we report the frequency of promising targets in advanced BTC currently under investigation in other advanced solid tumors, including KRAS G12C (1.0%), KRAS G12D (5.1%), PIK3CA mutations (6.8%), and ERBB2 amplifications (4.9%). CONCLUSIONS: These findings from the largest and most comprehensive study to date of cfDNA from patients with advanced BTC highlight the utility of cfDNA analysis in current management of this disease. Characterization of oncogenic drivers and mechanisms of therapeutic resistance in this study will inform drug development efforts to reduce mortality for patients with BTC.

Biochemical markers associated with executive function in adolescents with early and continuously treated phenylketonuria.

Debate continues as to why executive function (EF) continues to show impairments in children with early and continuously treated phenylketonuria (ECT-PKU). Using a mixed model, we measured EF in 10 adolescent children with ECT-PKU and 6 sibling controls, and examined associations between EF and (1) phenylalanine (phe) and (2) the phenylalanine : tyrosine ratio (phe : tyr). Measurements were taken on two occasions anticipated to yield variation in concurrent biochemistry resulting from changes in dietary compliance (i.e. holiday vs non-holiday period). A repeated measures anova using the Behaviour Rating Inventory of Executive Function yielded the following results: no significant interactions; two significant group effects of substantially impaired working memory and initiation skills in children with ECT-PKU compared with controls, irrespective of occasion and two significant time effects, suggestive of slightly poorer non-holiday planning and organization scores in both groups. Further analyses revealed that phe levels were not significantly different on these occasions, suggesting that holiday dietary compliance may be better than expected. Correlations between EF and biochemistry in children with PKU showed that participant's lifetime phe : tyr ratio was positively and significantly associated with EF impairment, more so than phe-only measures.

European Association of Urology - 32nd Annual Congress (March 24-28, 2017 - London, UK).

The European Association of Urology (EAU) Congress is Europe's biggest urological event and this year's meeting, in London, UK, brought together more than 13,000 participants from over 100 countries to discuss the latest research in this field. With 5 days' worth of lectures, debates, learning courses, presentations and live surgeries the congress provided plenty of opportunity to learn from the 1,400 experts presenting, as well as to network with international peers.

Executive function in adolescents with PKU and their siblings: Associations with biochemistry.

Previous research shows consistent and marked executive function impairment in children with early and continuously treated phenylketonuria. This between groups analysis (phenylketonuria group vs sibling controls) found no significant differences in executive function (although adolescents with phenylketonuria performed slightly worse than their siblings). Biochemical relationships with executive function were confined to long-term measures of high phenylalanine:tyrosine ratio exposure, as well as tyrosine exposure independent of phenylalanine. This study suggests that early and continuously treated PKU results in non-significant EF differences (compared to siblings), although the influence of long-term exposure to poorer metabolic control is still evident.

A reperfusion interval reduces the contractile deficit in skeletal muscle following tourniquet ischemia.

Bloodless surgical procedures on the extremities are achieved by application of a pneumatic tourniquet. The ischemia produced has deleterious effects on nerve and muscle function. It has been suggested that temporary interruption of ischemia by a reperfusion interval can prevent muscle and nerve injury. We investigated the muscle and nerve response to 3 hours of tourniquet ischemia, with and without a reperfusion interval after the first 2 hours of application, in a rodent model. Morphometric, contractile, and histologic parameters were measured. Tourniquet ischemia, with and without a reperfusion interval, results in muscle injury and a transient depression of muscle function. Introduction of a reperfusion interval reduces the severity of injury and increases the early rate of recovery. However, the later stages of recovery appear to be unaffected by reperfusion.

The effect of ischemic preconditioning on the recovery of skeletal muscle following tourniquet ischemia.

It has been well documented that ischemic preconditioning limits ischemic-reperfusion injury in cardiac muscle, but the ability of ischemic preconditioning to limit skeletal muscle injury is less clear. Previous reports have emphasized the beneficial effects of ischemic preconditioning on skeletal muscle structure and capillary perfusion but have not evaluated muscle function. We investigated the morphologic and functional consequences of ischemic preconditioning, followed by a 2-hour period of tourniquet ischemia on muscles in the rat hindlimb. The 2-hour ischemia was imposed without preconditioning, or was preceded by three brief (10 minutes on/10 minutes off) preischemic conditioning intervals. We compared muscle morphology, isometric contractile function, and muscle fatigue properties in predominantly fast-twitch, tibialis anterior muscles 3 (n = 8) and 7 (n = 8) days after ischemia-reperfusion. Two hours of ischemia, followed by reperfusion, results in a 20 percent reduction of muscle mass (p < 0.05) and a 33 percent reduction in tetanic tension (p < 0.05) when compared with controls (n = 8) at 3 days. The same protocol, when preceded by ischemic preconditioning, results in similar decreases in muscle mass and contractile function. Neuromuscular transmission was also impaired in both ischemic groups 7 days after ischemia. Nerve-evoked maximum tetanic tension was 69 percent of the tension produced by direct muscle stimulation in the ischemia group and 65 percent of direct tension in the ischemic preconditioning/ischemia group. In summary, ischemic preconditioning, using the same protocol reported to be effective in limiting infarct size in porcine muscle, had no significant benefit in limiting injury or improving recovery in the ischemic rat tibialis anterior. The value of ischemic preconditioning in reducing imposed ischemic-reperfusion-induced functional deficits in skeletal muscle remains to be demonstrated.

Wrongful life actions: the legal and ethical hurdles.

This article discusses the various legal and ethical issues arising out of the cause of action for wrongful life. This action involves a claim by a child that but for the negligence of the doctor, hospital or other medical institution, his or her mother would have terminated the pregnancy and he or she would not have been born. The courts have generally rejected this cause of action on the basis of legal, ethical and policy considerations. The author proposes that the legal hurdles can be overcome and that the ethical and policy considerations do not outweigh the desirability of upholding wrongful life claims.

Peripheral nerve and muscle function in the aging Fischer 344/brown-Norway rat.

This study was designed to define the age-related changes that occur in the F1 cross of the male Fischer 344 and brown Norway rats and to determine if these findings were associated with electrophysiologic abnormalities indicative of motor neuron loss. Contractility, morphologic, and histochemical studies were performed on the tibialis anterior muscles (TA) from 25 male rats at ages 6, 18 and 30-32 months. Tibialis anterior weight was 17% greater in the 18-mo vs 6-mo-old animals, but at age 32 months mean TA weight was 20% less than at 18 months. Other changes at 32 months included a 12% decrease in specific tension and reduced contractile/relaxation velocities of isometric twitches and maximal tetanic tension; findings associated with a 40% decrease in type IIb fiber cross-sectional area. Electrophysiologic studies on 15 rats revealed prolonged H-reflex latencies at 18 and 32 months. Needle electromyography demonstrated abnormal spontaneous activity consistent with peripheral axonal, not motor neuron loss. These findings demonstrate age-related changes in muscle mass and strength that are associated with changes in the peripheral nervous system. These findings are consistent with previous work in homozygous, inbred strains and help to establish the F1 cross of the Fischer 344 and brown Norway strains as a potentially useful rodent model in gerontologic studies of the neuromuscular system.

Bacterial suicide through stress.

Outside of the laboratory, bacterial cells are constantly exposed to stressful conditions, and an ability to resist those stresses is essential to their survival. However, the degree of stress required to bring about cell death varies with growth phase, amongst other parameters. Exponential phase cells are significantly more sensitive to stress than stationary phase ones, and a novel hypothesis has recently been advanced to explain this difference in sensitivity, the suicide response. Essentially, the suicide response predicts that rapidly growing and respiring bacterial cells will suffer growth arrest when subjected to relatively mild stresses, but their metabolism will continue: a burst of free-radical production results from this uncoupling of growth from metabolism, and it is this free-radical burst that is lethal to the cells, rather than the stress per se. The suicide response hypothesis unifies a variety of previously unrelated empirical observations, for instance induction of superoxide dismutase by heat shock, alkyl-hydroperoxide reductase by osmotic shock and catalase by ethanol shock. The suicide response also has major implications for current [food] processing methods.

A competitive microflora increases the resistance of Salmonella typhimurium to inimical processes: evidence for a suicide response.

The presence of a viable competitive microflora at cell densities of 10(8) CFU ml-1 protects an underlying population of 10(5) CFU of Salmonella typhimurium ml-1 against freeze injury. The mechanism of enhanced resistance was initially postulated to be via an RpoS-mediated adaptive response. By using an spvRA:: luxCDABE reporter we have shown that although the onset of RpoS-mediated gene expression was brought forward by the addition of a competitive microflora, the time taken for induction was measured in hours. Since the protective effect of a competitive microflora is essentially instantaneous, the stationary-phase adaptive response is excluded as the physiological mechanism. The only instantaneous effect of the competitive microflora was a reduction in the percent saturation of oxygen from 100% to less than 10%. For both mild heat treatment (55 degrees C) and freeze injury this change in oxygen tension affords Salmonella a substantive (2 orders of magnitude) enhancement in survival. By reducing the levels of dissolved oxygen through active respiration, a competitive microflora reduces oxidative damage to exponential-phase cells irrespective of the inimical treatment. These results have led us to propose a suicide hypothesis for the destruction of rapidly growing cells by inimical processes. In essence, the suicide hypothesis proposes that a mild inimical process leads to the growth arrest of exponential-phase cells and to the decoupling of anabolic and catabolic metabolism. The result of this is a free radical burst which is lethal to unadapted cells.

Metabolic fluctuation during a muscle contraction cycle.

Gated 31P-nuclear magnetic resonance followed the metabolic fluctuation in rat gastrocnemius muscle during a contraction cycle. Within 16 ms after stimulation, the phosphocreatine (PCr) level drops 11.3% from its reference state. The PCr minimum corresponds closely to the time of maximum force contraction. Pi increases stoichiometrically, while ATP remains constant. During a twitch, PCr hydrolysis produces 3.1 mumol ATP/g tissue, which is substantially higher than the reported 0.3 mumol ATP.twitch-1.g tissue-1 derived from steady-state experiments. The results reveal that a substantial energy fluctuation accompanies a muscle twitch.

Sonographic fetal growth standards. Are current data applicable to a racially mixed population?

Currently available sonographic growth standards for fetal head size, abdominal size, and limb length are based primarily on studies from white populations. To determine whether these published standards are appropriate for a racially mixed, indigent population, we compared our published data from a middle-class white population with data generated from a black/Hispanic population seen at a county hospital in Houston, Texas. No statistically significant differences were found for any of the following fetal sonographic parameters (20 to 41 weeks): biparietal diameter, head circumference, abdominal circumference, femur length.

High-repetitive submaximal treadmill exercise training: effect on normal and dystrophic mice.

Exercise as a treatment for muscular dystrophy is controversial. Whereas vigorous high-resistive exercise increases skeletal muscle degeneration in animals with neuromuscular disorders, the effect of low-intensity, high-repetitive exercise has been conflicting. The purpose of this study was to determine if low-intensity, high-repetitive exercise has a beneficial effect on dystrophic mice. Dystrophy mice and unaffected littermates were exercised daily starting at age three weeks on a treadmill (4 m/min, 18 degrees incline, 100 meters, three weeks' duration). Exercise increased the dystrophic soleus twitch tension, the rate of twitch tension development, and the rate of twitch tension relaxation by 55%, 58%, and 48%, respectively (p less than .05). The twitch:tetanus ratio increased by 57% (p less than .05). Both the soleus and the extensor digitorum longus from the exercised dystrophic mice had significantly less degeneration (as shown by reduced internal nuclei, necrosis, fiber splitting, and moth-eaten fibers) than the nonexercised dystrophic mice (p less than .05). This study suggests that exercise training programs can be beneficial or at least not result in further muscle fiber degeneration if (1) the exercise program is started early in the course of the disease; (2) submaximal high-repetitive or even high-resistive exercise is used; and (3) the histopathologic degeneration is slowly progressive or in an arrested state.

Estimation of fetal weight with the use of head, body, and femur measurements--a prospective study.

In utero estimates of fetal weight were evaluated prospectively in 109 fetuses with the use of sonographic models developed in a previous study. This report confirms that the best in utero weight estimates result from the use of models based on measurements of head size, abdominal size, and femur length. Since the accuracy of these models (1 SD = 7.5%) is significantly better than those based on measurements of head and body (e.g., biparietal diameter, abdominal circumference), we recommend routine use of such models in obstetric sonography.

Contractile and EMG studies of murine myotonia (mto) and muscular dystrophy (dy/dy).

This report focuses on the myotonic (mto) mouse, an autosomal recessive neuromuscular mutant first described in 1982. Studies in vivo confirmed the presence of hindlimb rigidity during walking and typical myotonic electromyographic (EMG) discharges that persisted after nerve transection and complete neuromuscular blockade. Studies of the contractility of mto muscles in vitro revealed reduced peak isometric tetanic tension and greatly prolonged relaxation times. Tubocurarine did not affect tension parameters, but did antagonize the delayed relaxation in vitro. On the basis of EMG studies alone this mutant can accurately be described as myotonic. Reduction of the contractile abnormalities by tubocurarine in vitro, however, poses further questions regarding the nature of the disorder. Although the more familiar dystrophic mouse (dy/dy) has been termed "myotonic" by some, the new mto mutant differs from it in all aspects examined.

Estimating fetal age using multiple parameters: a prospective evaluation in a racially mixed population.

This study was designed to evaluate the accuracy of regression models for menstrual age prediction using single versus multiple fetal ultrasonographic measurements (biparietal diameter, head circumference, abdominal circumference, and femur length). The models tested had been previously developed from a study of middle-class white patients in a private hospital. The current study population consisted of 300 indigent black and Hispanic patients seen in a county hospital. This study demonstrated prospectively that the use of multiple parameters in estimating fetal age offers a significant advantage over any single parameter used alone and that the regression equations developed from a middle-class white population appear to be applicable to fetuses from a population with different socioeconomic and racial characteristics.

Fast and slow skeletal muscles: effect of ethanol on contractility of muscles from mice.

We examined in vitro the effect of ethanol at four concentrations (0g%, 0.1g%, 0.2g%, and 0.4g%) on contractile parameters of 40 fast extensor digitorum longus (EDL) and 40 slow soleus muscles from healthy mice at 35C. Preparations were curarized to avoid the possible effect of ethanol on the terminal axons or skeletal neuromuscular junction. Contractile parameters measured included: (1) twitch and tetanic tension; (2) rate of tension development; (3) time to peak tension and half relaxation for twitch; (4) time to first evidence of relaxation in the tetanus; and (5) maximum rate of relaxation. The three lower concentrations of ethanol had no significant effect on muscle contractility; however, the 0.4g% dose reduced EDL twitch tension by 9%. High doses of ethanol (2.5g%) reduced the tetanic tension produced by the EDL and soleus muscles 31% and 26%, respectively. Ethanol at 2.5g% also reduced the twitch tension of the EDL and soleus by 50% and 38%, respectively. The data suggested that the 0.4g% is the highest dose of ethanol that should be used to dilute drugs in a solution that will bathe directly stimulated curarized muscle without confounding effects. In addition, it is highly unlikely that a direct effect of ethanol on muscle contractility in humans is related to an impairment in driving.

Effect of hind-limb suspension on young and adult skeletal muscle. I. Normal mice.

The purpose of this study was to determine the effect of hind-limb suspension (HS) on morphometric, histologic, and contractile characteristics of fast extensor digitorum longus (EDL) and slow soleus (SOL) twitch muscles in adult and immature mice. Hind-limb suspension for 2 weeks was used to produce atrophy in two groups of mice, ages 4 and 12 weeks, with nonsuspended animals serving as controls. Young HS mice exhibited marked decreases in SOL weight, length, cross-sectional area (CSA), twitch and tetanic tensions, and rates of tension development and relaxation, with increases in fatigue resistance. HS reduced the diameter of both type I and IIA fibers, increased the percentage of type I fibers, and decreased the percentage of type IIA fibers in both young and adult SOL. Muscle weight, length, CSA, IIA and IIB fiber areas, and maximum rate of tetanic tension development were decreased in EDL of young HS mice; fatigue resistance and EDL half-relaxation times were increased. For most parameters evaluated, slow twitch muscle was more affected than fast twitch. HS affected contractile characteristics less than morphometric or histologic parameters. Rates of tension development and relaxation were the contractile parameters most affected by HS, and the time parameters of contraction were least affected. For all measurements young mice were more affected than adult mice.