Real-world application of tumor mutational burden-high (TMB-high) and microsatellite instability (MSI) confirms their utility as immunotherapy biomarkers.

INTRODUCTION: Microsatellite instability (MSI) testing and tumor mutational burden (TMB) are genomic biomarkers used to identify patients who are likely to benefit from immune checkpoint inhibitors. Pembrolizumab was recently approved by the Food and Drug Administration for use in TMB-high (TMB-H) tumors, regardless of histology, based on KEYNOTE-158. The primary objective of this retrospective study was real-world applicability and use of immunotherapy in TMB/MSI-high patients to lend credence to and refine this biomarker. METHODS: Charts of patients with advanced solid tumors who had MSI/TMB status determined by next generation sequencing (NGS) (FoundationOne CDx) were reviewed. Demographics, diagnosis, treatment history, and overall response rate (ORR) were abstracted. Progression-free survival (PFS) was determined from Kaplan-Meier curves. PFS1 (chemotherapy PFS) and PFS2 (immunotherapy PFS) were determined for patients who received immunotherapy after progressing on chemotherapy. The median PFS2/PFS1 ratio was recorded. RESULTS: MSI-high or TMB-H [≥20 mutations per megabase (mut/MB)] was detected in 157 adults with a total of 27 distinct tumor histologies. Median turnaround time for NGS was 73 days. ORR for most recent chemotherapy was 34.4%. ORR for immunotherapy was 55.9%. Median PFS for patients who received chemotherapy versus immunotherapy was 6.75 months (95% confidence interval, 3.9-10.9 months) and 24.2 months (95% confidence interval, 9.6 months to not reached), respectively (P = 0.042). Median PFS2/PFS1 ratio was 4.7 in favor of immunotherapy. CONCLUSION: This real-world study reinforces the use of TMB as a predictive biomarker. Barriers exist to the timely implementation of NGS-based biomarkers and more data are needed to raise awareness about the clinical utility of TMB. Clinicians should consider treating TMB-H patients with immunotherapy regardless of their histology.

Adjuvant chemotherapy for elderly patients with stage I non-small-cell lung cancer ≥4 cm in size: an SEER-Medicare analysis.

BACKGROUND: The role of adjuvant chemotherapy for non-small-cell lung cancer (NSCLC) stage I patients with tumors size ≥4 cm is not well established in the elderly. PATIENTS AND METHODS: We identified 3289 patients with stage I NSCLC (T2N0M0 and tumor size ≥4 cm) who underwent lobectomy from the Surveillance, Epidemiology and End Results (SEER)-Medicare linked database diagnosed from 1992 to 2009. Overall survival and rates of serious adverse events (defined as those requiring admission to hospital) were compared between patients treated with resection alone, platinum-based adjuvant chemotherapy, or postoperative radiation (PORT) with or without adjuvant chemotherapy. Propensity scores for receiving each treatment were calculated and survival analyses were conducted using inverse probability weights based on the propensity score. RESULTS: Overall, 84% patients were treated with resection alone, 9% received platinum-based adjuvant chemotherapy, and 7% underwent PORT with or without adjuvant chemotherapy. Adjusted analysis showed that adjuvant chemotherapy [hazard ratio (HR), 0.82; 95% confidence interval (CI) 0.68-0.98] was associated with improved survival compared with resection alone. Conversely, the use of PORT with or without adjuvant chemotherapy (HR 1.91; 95% CI 1.64-2.23) was associated with worse outcomes. Patients receiving adjuvant chemotherapy had more serious adverse events compared with those treated with resection alone, with neutropenia (odds ratio, 21.2; 95% CI 5.8-76.6) being most significant. No significant difference was observed in rates of fever, cytopenias, nausea, and renal dysfunction. CONCLUSIONS: Platinum-based adjuvant chemotherapy is associated with reduced mortality and increased serious adverse events in elderly patients with stage I NSCLC and tumor size ≥4 cm.

British and Canadian views on the ethics of paediatric clinical trials.

BACKGROUND: Ethical problems are quoted as a reason not to perform clinical trials in children. Little is known about the views of researchers regarding ethics. OBJECTIVES: A pilot study was conducted to assess the applicability of a questionnaire design containing trial scenarios to examine views regarding the use of children in drug trials and to elicit possible international differences. SETTING: Paediatricians and researchers in the United Kingdom and Canada. METHODS: Responders were presented with a questionnaire containing direct questions and six trial scenarios, each containing an ethical dilemma. Responders were asked regarding their own approval and their perceived opinion of whether an ethical review board (ERB) would approve. RESULTS: One hundred questionnaires (50 each country) were received. Few responders had research ethics training (14% United Kingdom and 8% Canada). Most (80 and 88%) felt children could be harmed by participation in trials and half (47 and 59%) felt children should only participate if they receive direct benefit. Many (58 and 61%) disagreed with payments beyond travel expenses. In the trial scenarios, 34% of responders were willing to enter healthy children in a pharmacokinetics study of an antibiotic for cystic fibrosis and 22% considered their ERBs would approve. Only a third (33%) would enter children in an analgesia trial that was placebo-controlled. CONCLUSION: Using healthy children and placebos in trials caused concern. Similar views were found between the two countries. The majority had no training in research ethics. The study highlights the usefulness of a questionnaire with clinical trial scenarios to try to elicit views on the ethics of conducting research in children.

Contactin associates with Na+ channels and increases their functional expression.

Contactin (also known as F3, F11) is a surface glycoprotein that has significant homology with the beta2 subunit of voltage-gated Na(+) channels. Contactin and Na(+) channels can be reciprocally coimmunoprecipitated from brain homogenates, indicating association within a complex. Cells cotransfected with Na(+) channel Na(v)1.2alpha and beta1 subunits and contactin have threefold to fourfold higher peak Na(+) currents than cells with Na(v)1.2alpha alone, Na(v)1.2/beta1, Na(v)1.2/contactin, or Na(v)1.2/beta1/beta2. These cells also have a correspondingly higher saxitoxin binding, suggesting an increased Na(+) channel surface membrane density. Coimmunoprecipitation of different subunits from cell lines shows that contactin interacts specifically with the beta1 subunit. In the PNS, immunocytochemical studies show a transient colocalization of contactin and Na(+) channels at new nodes of Ranvier forming during remyelination. In the CNS, there is a particularly high level of colocalization of Na(+) channels and contactin at nodes both during development and in the adult. Contactin may thus significantly influence the functional expression and distribution of Na(+) channels in neurons.

The intracellular segment of the sodium channel beta 1 subunit is required for its efficient association with the channel alpha subunit.

Sodium channels consist of a pore-forming alpha subunit and auxiliary beta 1 and beta 2 subunits. The subunit beta 1 alters the kinetics and voltage-dependence of sodium channels expressed in Xenopus oocytes or mammalian cells. Functional modulation in oocytes depends on specific regions in the N-terminal extracellular domain of beta 1, but does not require the intracellular C-terminal domain. Functional modulation is qualitatively different in mammalian cells, and thus could involve different molecular mechanisms. As a first step toward testing this hypothesis, we examined modulation of brain Na(V)1.2a sodium channel alpha subunits expressed in Chinese hamster lung cells by a mutant beta1 construct with 34 amino acids deleted from the C-terminus. This deletion mutation did not modulate sodium channel function in this cell system. Co-immunoprecipitation data suggest that this loss of functional modulation was caused by inefficient association of the mutant beta 1 with alpha, despite high levels of expression of the mutant protein. In Xenopus oocytes, injection of approximately 10,000 times more mutant beta 1 RNA was required to achieve the level of functional modulation observed with injection of full-length beta 1. Together, these findings suggest that the C-terminal cytoplasmic domain of beta 1 is an important determinant of beta1 binding to the sodium channel alpha subunit in both mammalian cells and Xenopus oocytes.

Characterization of sodium channel alpha- and beta-subunits in rat and mouse cardiac myocytes.

BACKGROUND: Sodium channels isolated from mammalian brain are composed of alpha-, beta(1)-, and beta(2)-subunits. The composition of sodium channels in cardiac muscle, however, has not been defined, and disagreement exists over which beta-subunits are expressed in the myocytes. Some investigators have demonstrated beta(1) expression in heart. Others have not detected any auxiliary subunits. On the basis of Northern blot analysis of total RNA, beta(2) expression has been thought to be exclusive to neurons and absent from cardiac muscle. METHODS AND RESULTS: The goal of this study was to define the subunit composition of cardiac sodium channels in myocytes. We show that cardiac sodium channels are composed of alpha-, beta(1)-, and beta(2)-subunits. Nav1.5 and Nav1.1 are expressed in myocytes and are associated with beta(1)- and beta(2)-subunits. Immunocytochemical localization of Nav1.1, beta(1), and beta(2) in adult heart sections showed that these subunits are expressed at the Z lines, as shown previously for Nav1.5. Coexpression of Nav1.5 with beta(2) in transfected cells resulted in no detectable changes in sodium current. CONCLUSIONS: Cardiac sodium channels are composed of alpha- (Nav1.1 or Nav1.5), beta(1)-, and beta(2)-subunits. Although beta(1)-subunits modulate cardiac sodium channel current, beta(2)-subunit function in heart may be limited to cell adhesion.

Antiepileptic drug therapy in the twenty first century.

In the last 25 years, particularly the last decade, there have been many advances relating to all aspects of epilepsy i.e. pathophysiology, diagnosis, pharmacotherapy and surgical interventions. Noteworthy has been the progress in terms of understanding of the established antiepileptic drugs (AEDs) and introduction of several newer agents developed rationally, on the basis of now available information on the biochemical changes in the epileptic brain. Data is accumulating regarding the use of newer agents but they still need to stand the test of time. Many of the newer AEDs may offer a better tolerability because of favorable pharmacokinetic characteristics and minimal drug interactions. However, serious adverse events have been associated with felbamate and lamotrigine already and for other newer agents reliable and accurate data needs to be generated.

The L1-type cell adhesion molecule neuroglian influences the stability of neural ankyrin in the Drosophila embryo but not its axonal localization.

Ankyrins are linker proteins, which connect various membrane proteins, including members of the L1 family of neural cell adhesion molecules, with the submembranous actin-spectrin skeleton. Here we report the cloning and characterization of a second, novel Drosophila ankyrin gene (Dank2) that appears to be the result of a gene duplication event during arthropod evolution. The Drosophila L1-type protein neuroglian interacts with products from both Drosophila ankyrin genes. Whereas the previously described ankyrin gene is ubiquitously expressed during embryogenesis, the expression of Dank2 is restricted to the nervous system in the Drosophila embryo. The absence of neuroglian protein in a neuroglian null mutant line causes decreased levels of Dank2 protein in most neuronal cells. This suggests that neuroglian is important for the stability of Dank2 protein. However, neuroglian is not required for Dank2 axonal localization. In temperature-sensitive neuroglian mutants in which neuroglian protein is mislocated at the restrictive temperature to an intracellular location in the neuronal soma, Dank2 protein can still be detected along embryonic nerve tracts.

Dopaminergic involvement in adenosine A1 receptor-mediated antinociception in the tail flick latency model in mice.

The interaction between the adenosinergic and dopaminergic systems in nociception was assessed in the tail flick latency (TFL) test in mice. Adenosine exhibited qualitatively different responses depending on the dose: Adenosine 10 and 100 mg/kg i.p. caused antinociception as evidenced by an increase in TFL while the middle dose of 30 mg/kg decreased TFL. On the other hand, the specific adenosine A1 receptor agonist N6-cyclopentyladenosine (CPA) at doses of 0.05, 0.1, 0.25 and 0.5 mg/kg (i.p.) resulted in dose-dependent antinociception. The antinociceptive effect of CPA was reversed by classical albeit nonspecific adenosine receptor antagonist theophylline (5 mg/kg) at a dose which had no effect on TFL per se. A low dose (1 mg/kg i.p.) of the dopaminergic agonist apomorphine caused an early mild hyperalgesic response while the high dose (10 mg/kg i.p.) had no significant effect on TFL. The antinociceptive effect of CPA was attenuated by pretreatment with low dose apomorphine while pretreatment with the high dose caused mild but insignificant potentiation. Theophylline, when administered prior to apomorphine failed to modify the nociceptive response. The results suggest that an interaction between adenosine and dopamine may be involved in nociception.

Sodium channel beta subunits mediate homophilic cell adhesion and recruit ankyrin to points of cell-cell contact.

Sodium channels isolated from mammalian brain are composed of alpha, beta1, and beta2 subunits. The auxiliary beta subunits do not form the ion conducting pore, yet play important roles in channel modulation and plasma membrane expression. beta1 and beta2 are transmembrane proteins with one extracellular V-set immunoglobulin (Ig) protein domain. It has been shown recently that beta1 and beta2 interact with the extracellular matrix proteins tenascin-C and tenascin-R. In the present study we show that rat brain beta1 and beta2, but not alphaIIA, subunits interact in a trans-homophilic fashion, resulting in recruitment of the cytoskeletal protein ankyrin to sites of cell-cell contact in transfected Drosophila S2 cells. Whereas alphaIIA subunits expressed alone do not cause cellular aggregation, beta subunits co-expressed with alphaIIA retain the ability to adhere and recruit ankyrin. Truncated beta subunits lacking cytoplasmic domains interact homophilically to produce cell aggregation but do not recruit ankyrin. Thus, the cytoplasmic domains of beta1 and beta2 are required for cytoskeletal interactions. It is hypothesized that sodium channel beta subunits serve as a critical communication link between the extracellular and intracellular environments of the neuron and may play a role in sodium channel placement at nodes of Ranvier.

Tenascin-R is a functional modulator of sodium channel beta subunits.

Voltage-gated sodium channels isolated from mammalian brain are composed of alpha, beta1, and beta2 subunits. The alpha subunit forms the ion conducting pore of the channel, whereas the beta1 and beta2 subunits modulate channel function, as well as channel plasma membrane expression levels. beta1 and beta2 each contain a single, extracellular Ig-like domain with structural similarity to the neural cell adhesion molecule (CAM), myelin Po. beta2 contains strong amino acid homology to the third Ig domain and to the juxtamembrane region of F3/contactin. Many CAMs of the Ig superfamily have been shown to interact with extracellular matrix molecules. We hypothesized that beta2 may interact with tenascin-R (TN-R), an extracellular matrix molecule that is secreted by oligodendrocytes during myelination and that binds F3-contactin. We show here that cells expressing sodium channel beta1 or beta2 subunits are functionally modulated by TN-R. Transfected cells stably expressing beta1 or beta2 subunits initially recognized and then were repelled from TN-R substrates. The cysteine-rich amino-terminal domain of TN-R expressed as a recombinant peptide, termed EGF-L, appears to be responsible for the repellent effect on beta subunit-expressing cells. The epidermal growth factor-like repeats and fibronectin-like repeats 6-8 are most effective in the initial adhesion of beta subunit-expressing cells. Application of EGF-L to alphaIIAbeta1beta2 channels expressed in Xenopus oocytes potentiated expressed sodium currents without significantly altering current time course or the voltage dependence of current activation or inactivation. Thus, sodium channel beta subunits appear to function as CAMs, and TN-R may be an important regulator of sodium channel localization and function in neurons.

Effect of adenosinergic modulation on the anticonvulsant effect of phenobarbitone and carbamazepine.

Adenosine has been demonstrated to have an anticonvulsant action which is mediated predominantly by the adenosine A1 receptor subtype. The present study was conducted to determine if the adenosinergic system and adenosine A1 receptors are involved in the anticonvulsant action of the antiepileptic drugs phenobarbitone and carbamazepine, in pentylenetetrazole (PTZ)-induced seizures in rats. The specific adenosine A1 receptor antagonist, DPCPX (1 mg/kg i.p.), had no effect on the anticonvulsant action of the two antiepileptic drugs. However, the nonspecific adenosine receptor antagonist, theophylline (50 and 100 mg/kg i.p.), reversed the anticonvulsant action of carbamazepine completely and that of phenobarbitone partially. This suggests that adenosine A1 receptors do not mediate the anticonvulsant effects of these agents. When phenobarbitone/carbamazepine were coadministered with adenosine/N6-cyclopentyladenosine (CPA), a specific adenosine A1 receptor agonist, an enhancement in protection against PTZ-induced seizures was observed. The diversity of anticonvulsant mechanism of carbamazepine/phenobarbitone and that of adenosinergic agents could be responsible for this effect.

Effect of different lithium priming schedule on pilocarpine-induced status epilepticus in rats.

The conventional method of inducing status epilepticus by injecting pilocarpine 24 h after lithium chloride (LiCl) administration lacks kinetic correlate. Therefore, the present study was conducted to see the effect of altering the time schedule of lithium administration (from 2 to 72 h) on the convulsive behavior in response to pilocarpine challenge in rats. It was observed that reducing the pretreatment time of lithium from 24 to 2 h produced convulsions in 100% of the rats. However, incidence of convulsions decreased when lithium pretreatment time was increased to 48 h and no convulsions were recorded with 72-h lithium time schedule. The neuronal damage assessed histologically and the effectiveness of diazepam was similar irrespective of time schedule. This study provides evidence that LiCl pretreatment schedule can be adopted at any time between 2 to 24 h to suit the convenience and objectives of the experiment.

METHODS AND CONSIDERATIONS FOR EXPERIMENTAL EVALUATION OF ANTIEPILEPTIC DRUGS.

Research into epilepsy and development of antiepileptic drugs relies heavily on studies in experimental animals. Though the conventional pentylenetetrazole-induced seizures and electroshock-induced seizures remain the mainstay of any antiepileptic drug screening protocol, considering the diversity of seizure types and underlying pathologies encountered in epilectics, numerous other seizure models have been developed. Some of these experimental models of seizures and considerations governing their selection are reviewed.

Cis-activation of L1-mediated ankyrin recruitment by TAG-1 homophilic cell adhesion.

Neural cell adhesion molecules (CAMs) of the immunoglobulin (Ig) superfamily mediate not only cell aggregation but also growth cone guidance and neurite outgrowth. In this study we demonstrate that two neural CAMs, L1-CAM and TAG-1, induce the homophilic aggregation of Drosophila S2 cells but are unable to interact with each other when expressed on different cells (trans-interaction). However, immunoprecipitations from cells co-expressing L1-CAM and TAG-1 showed a strong cis-interaction between the two molecules in the plane of the plasma membrane. TAG-1 is linked to the membrane by a glycosylphosphatidylinositol (GPI) anchor and therefore is unable to directly interact with cytoplasmic proteins. In contrast, L1-CAM-mediated homophilic cell adhesion induces the selective recruitment of the membrane skeleton protein ankyrin to areas of cell contact. Immunolabeling experiments in which S2 cells expressing TAG-1 were mixed with cells co-expressing L1-CAM and TAG-1 demonstrated that the homophilic interaction between TAG-1 molecules results in the cis-activation of L1-CAM to bind ankyrin. This TAG-1-dependent recruitment of the membrane skeleton provides an example of how GPI-anchored CAMs are able to transduce signals to the cytoplasm. Furthermore, such interactions might ultimately result in the recruitment and the activation of other signaling molecules at sites of cell contacts.

Structural requirements for outside-in and inside-out signaling by Drosophila neuroglian, a member of the L1 family of cell adhesion molecules.

Expression of the Drosophila cell adhesion molecule neuroglian in S2 cells leads to cell aggregation and the intracellular recruitment of ankyrin to cell contact sites. We localized the region of neuroglian that interacts with ankyrin and investigated the mechanism that limits this interaction to cell contact sites. Yeast two-hybrid analysis and expression of neuroglian deletion constructs in S2 cells identified a conserved 36-amino acid sequence that is required for ankyrin binding. Mutation of a conserved tyrosine residue within this region reduced ankyrin binding and extracellular adhesion. However, residual recruitment of ankyrin by this mutant neuroglian molecule was still limited to cell contacts, indicating that the lack of ankyrin binding at noncontact sites is not caused by tyrosine phosphorylation. A chimeric molecule, in which the extracellular domain of neuroglian was replaced with the corresponding domain from the adhesion molecule fasciclin II, also selectively recruited ankyrin to cell contacts. Thus, outside-in signaling by neuroglian in S2 cells depends on extracellular adhesion, but does not depend on any unique property of its extracellular domain. We propose that the recruitment of ankyrin to cell contact sites depends on a physical rearrangement of neuroglian in response to cell adhesion, and that ankyrin binding plays a reciprocal role in stabilizing the adhesive interaction.

Influence of adenosine agonists and antiepileptic drugs on theophylline-induced seizures in rats.

Seizures is a major toxicity of theophylline. The mechanism of theophylline-induced seizures is not known, but antagonism at adenosine receptors may be a possibility. The effect of pretreatment with different doses of adenosine (100, 500 and 1000 mg/kg, i.p.), and the adenosine A1 receptor agonist N6-cyclopentyladenosine (CPA), 1, 5 and 10 mg/kg, i.p., was studied against seizures induced by theophylline in rats. Both these drugs, at all dose levels tested, failed to protect theophylline seizures. Thus adenosinergic system is unlikely to be involved in mediating the convulsant action of theophylline. On the other hand, the conventional antiepileptic drugs, i.e. diazepam (4 mg/kg), sodium valproate (300 mg/kg) and phenobarbitone (50 mg/kg), but not carbamazepine, afforded some protection. The modification of course of seizures, by the antiepileptic drugs suggests the involvement of some other alternate mechanism in theophylline-induced seizures.

Effect of theophylline on diazepam and sodium valproate protection in pentylenetetrazole - kindled seizures in rats.

Theophylline is well known for its convulsant and proconvulsant action. Some experimental studies also suggest that theophylline and other methylxanthines may impair the protection of antiepileptic drugs. The interaction of theophylline and the antiepileptic drugs diazepam and sodium valproate was studied in pentylenetetrazole (PTZ) - kindled seizures in rats. Pretreatment with both diazepam 4 mg/kg and sodium valproate 300 mg/kg, i.p., showed protection against PTZ kindled seizures. Theophylline, 50 mg/kg, i.p., when given before the antiepileptic drugs, failed to reverse their protection. Since theophylline has an adenosine receptor antagonist activity which may be responsible for its convulsant potential, the results indicate non-involvement of adenosinergic mechanisms in the mechanisms of actions of these antiepileptic drugs.

A simple device for recording seizure activity in rats.

Experimental research on epilepsy and antiepileptic drugs in rodents utilizes different models (e.g., chemical, electrical or genetic) which have different end points for evaluating seizure activity. To overcome the possibility of observer bias and the limitation of nonavailability of a hard copy of seizure activity, a simple method is described. A horizontal perspex platform is mounted on four springs of suitable tension inside a perspex chamber. A force transducer is attached to the platform to record seizure activity on paper by a physiograph. Using this assembly, the incidence, latency and duration of different seizure components, i.e., myoclonic jerks and generalized clonic seizures, after pentyleneterrazole administration (60 mg/kg i.p.) in rats were recorded. The effects of pretreatment with anticonvulsant doses of diazepam (4 mg/kg i.p.) and sodium valproate (300 mg/kg i.p.) were studied. Chemical kindling was induced by repeated, alternate-day injections of a subconvulsant dose of pentylenetetrazole (30 mg/kg i.p.) for 10 weeks and seizure activity during different stages of development of kindling was recorded. The described method not only successfully records the different seizure components but also quantifies the latency and duration of seizure activity.