Biological and technical factors in the assessment of blood-based tumor mutational burden (bTMB) in patients with NSCLC.

BACKGROUND: Patients treated with immunotherapy are at risk of considerable adverse events, and the ongoing struggle is to accurately identify the subset of patients who will benefit. Tumor mutational burden (TMB) has emerged as a promising predictive biomarker but requires tumor tissue which is not always available. Blood-based TMB (bTMB) may provide a minimally invasive assessment of mutational load. However, because of the required sequencing depth, bTMB analysis is costly and prone to false negative results. This study attempted to design a minimally sized bTMB panel, examined a counting-based method for bTMB in patients with stage I to IV non-small cell lung cancer (NSCLC) and evaluated both technical factors such as bTMB and tissue-based TMB (tTMB) cut-off, as well as sample-related factors such as cell-free DNA input mass which influence the correlation between bTMB and tTMB. METHODS: Tissue, plasma, and whole blood samples collected as part of the LEMA trial (NCT02894853) were used in this study. Samples of 185 treatment naïve patients with stage I to IV NSCLC were sequenced at the Roche Sequencing Solutions with a custom panel designed for TMB, using reagents and workflows derived from the AVENIO Tumor Tissue and circulating tumor DNA Analysis Kits. RESULTS: A TMB panel of 1.1 Mb demonstrated highly accurate TMB high calls with a positive predictive value of 95% when using a tTMB cut-off of 16 mut/Mb, corresponding with 42 mut/Mb for bTMB. The positive per cent agreement (PPA) of bTMB was relatively low at 32%. In stage IV samples with at least 20 ng of cfDNA input, PPA of bTMB improved to 63% and minimizing the panel to a subset of 577 kb was possible while maintaining 63% PPA. CONCLUSION: Plasma samples with high bTMB values are highly correspondent with tTMB, whereas bTMB low results may also be the result of low tumor burden at earlier stages of disease as well as poorly shedding tumors. For advanced stages of disease, PPA (sensitivity) of bTMB is satisfactory in comparison to tTMB, even when using a panel of less than 600 kb, warranting consideration of bTMB as a predictive biomarker for patients with NSCLC eligible for immunotherapy in the future.

Implementation of Novel Molecular Biomarkers for Non-small Cell Lung Cancer in the Netherlands: How to Deal With Increasing Complexity.

The diagnostic landscape of non-small cell lung cancer (NSCLC) is changing rapidly with the availability of novel treatments. Despite high-level healthcare in the Netherlands, not all patients with NSCLC are tested with the currently relevant predictive tumor markers that are necessary for optimal decision-making for today's available targeted or immunotherapy. An expert workshop on the molecular diagnosis of NSCLC involving pulmonary oncologists, clinical chemists, pathologists, and clinical scientists in molecular pathology was held in the Netherlands on December 10, 2018. The aims of the workshop were to facilitate cross-disciplinary discussions regarding standards of practice, and address recent developments and associated challenges that impact future practice. This paper presents a summary of the discussions and consensus opinions of the workshop participants on the initial challenges of harmonization of the detection and clinical use of predictive markers of NSCLC. A key theme identified was the need for broader and active participation of all stakeholders involved in molecular diagnostic services for NSCLC, including healthcare professionals across all disciplines, the hospitals and clinics involved in service delivery, healthcare insurers, and industry groups involved in diagnostic and treatment innovations. Such collaboration is essential to integrate different technologies into molecular diagnostics practice, to increase nationwide patient access to novel technologies, and to ensure consensus-preferred biomarkers are tested.

Limited impact of Cntn4 mutation on autism-related traits in developing and adult C57BL/6J mice.

BACKGROUND: Mouse models offer an essential tool to unravel the impact of genetic mutations on autism-related phenotypes. The behavioral impact of some important candidate gene models for autism spectrum disorder (ASD) has not yet been studied, and existing characterizations mostly describe behavioral phenotypes at adult ages, disregarding the developmental nature of the disorder. In this context, the behavioral influence of CNTN4, one of the strongest suggested ASD candidate genes, is unknown. Here, we used our recently established developmental test battery to characterize the consequences of disruption of contactin 4 (Cntn4) on neurological, sensory, cognitive, and behavioral phenotypes across different developmental stages. METHODS: C57BL/6J mice with heterozygous and homozygous disruption of Cntn4 were studied through an extensive, partially longitudinal, test battery at various developmental stages, including various paradigms testing social and restricted repetitive behaviors. RESULTS: Developmental neurological and cognitive screenings revealed no significant differences between genotypes, and ASD-related behavioral domains were also unchanged in Cntn4-deficient versus wild-type mice. The impact of Cntn4-deficiency was found to be limited to increased startle responsiveness following auditory stimuli of different high amplitudes in heterozygous and homozygous Cntn4-deficient mice and enhanced acquisition in a spatial learning task in homozygous mice. CONCLUSIONS: Disruption of Cntn4 in the C57BL/6J background does not affect specific autism-related phenotypes in developing or adult mice but causes subtle non-disorder specific changes in sensory behavioral responses and cognitive performance.

A rodent version of the Iowa Gambling Task: 7 years of progress.

In the Iowa Gambling Task (IGT) subjects need to find a way to earn money in a context of variable wins and losses, conflicting short-term and long-term pay-off, and uncertainty of outcomes. In 2006, we published the first rodent version of the IGT (r-IGT; Behavior Research Methods 38, 470-478). Here, we discuss emerging ideas on the involvement of different prefrontal-striatal networks in task-progression in the r-IGT, as revealed by our studies thus far. The emotional system, encompassing, among others, the orbitofrontal cortex, infralimbic cortex and nucleus accumbens (shell and core area), may be involved in assessing and anticipating the value of different options in the early stages of the task, i.e., as animals explore and learn task contingencies. The cognitive control system, encompassing, among others, the prelimbic cortex and dorsomedial striatum, may be involved in instrumental goal-directed behavior in later stages of the task, i.e., as behavior toward long-term options is strengthened (reinforced) and behavior toward long-term poor options is weakened (punished). In addition, we suggest two directions for future research: (1) the role of the internal state of the subject in decision-making, and (2) studying differences in task-related costs. Overall, our studies have contributed to understanding the interaction between the emotional system and cognitive control system as crucial to navigating human and non-human animals alike through a world of variable wins and losses, conflicting short-term and long-term pay-offs, and uncertainty of outcomes.

Enhancing the value of psychiatric mouse models; differential expression of developmental behavioral and cognitive profiles in four inbred strains of mice.

The behavioral characterization of animal models of psychiatric disorders is often based upon independent traits measured at adult age. To model the neurodevelopmental aspects of psychiatric pathogenesis, we introduce a novel approach for a developmental behavioral analysis in mice. C57BL/6J (C57) mice were used as a reference strain and compared with 129S1/SvImJ (129Sv), BTBR T+tf/J (BTBR) and A/J (AJ) strains as marker strains for aberrant development. Mice were assessed at pre-adolescence (4 weeks), adolescence (6 weeks), early adulthood (8 weeks) and in adulthood (10-12 weeks) on a series of behavioral tasks measuring general health, neurological reflexes, locomotor activity, anxiety, short- and long-term memory and cognitive flexibility. Developmental delays in short-term object memory were associated with either a hypo-reactive profile in 129Sv mice or a hyper-reactive profile in BTBR mice. Furthermore, BTBR mice showed persistent high levels of repetitive grooming behavior during all developmental stages that was associated with the adult expression of cognitive rigidity. In addition, strain differences in development were observed in puberty onset, touch escape, and body position. These data showed that this longitudinal testing battery provides sufficient behavioral and cognitive resolution during different development stages and offers the opportunity to address the behavioral developmental trajectory in genetic mouse models for neurodevelopmental disorders. Furthermore, the data revealed that the assessment of multiple behavioral and cognitive domains at different developmental stages is critical to determine confounding factors (e.g., impaired motor behavior) that may interfere with the behavioral testing performance in mouse models for brain disorders.

A critical review of sex differences in decision-making tasks: focus on the Iowa Gambling Task.

It has been observed that men and women show performance differences in the Iowa Gambling Task (IGT), a task of decision-making in which subjects through exploration learn to differentiate long-term advantageous from long-term disadvantageous decks of cards: men choose more cards from the long-term advantageous decks than women within the standard number of 100 trials. Here, we aim at discussing psychological mechanisms and neurobiological substrates underlying sex differences in IGT-like decision-making. Our review suggests that women focus on both win-loss frequencies and long-term pay-off of decks, while men focus on long-term pay-off. Furthermore, women may be more sensitive to occasional losses in the long-term advantageous decks than men. As a consequence hereof, women need 40-60 trials in addition before they reach the same level of performance as men. These performance differences are related to differences in activity in the orbitofrontal cortex and dorsolateral prefrontal cortex as well as in serotonergic activity and left-right hemispheric activity. Sex differences in orbitofrontal cortex activity may be due to organisational effects of gonadal hormones early in life. The behavioural and neurobiological differences in the IGT between men and women are an expression of more general sex differences in the regulation of emotions. We discuss these findings in the context of sex differences in information processing related to evolutionary processes. Furthermore we discuss the relationship between these findings and real world decision-making.

Male and female Wistar rats differ in decision-making performance in a rodent version of the Iowa Gambling Task.

The Iowa Gambling Task (IGT) measures decision-making processes by simulating real-life decisions involving reward, punishment, and uncertainty of outcomes. In humans, men show more choices for the advantageous option than women. Here, we investigated sex differences in a rat model of the IGT (r-IGT). In our r-IGT mildly food-deprived rats learn to differentiate a long-term advantageous arm from a long-term disadvantageous arm differing in frequency and amount of sugar pellets as well as unpalatable but not uneatable quinine-treated sugar pellets. We also used a T-maze discrimination procedure in which rats learn to differentiate a high from a low reward arm to further explore sex differences in reward-related decision-making. In line with human data, male rats showed a stronger task progression of choices for the advantageous option than female rats. Furthermore, male rats showed more win-stay and less lose-shift behaviour in the advantageous arm as the task progressed than female rats. Whilst both male and female rats had a stronger preference for the high over the small reward arm in the T-maze, males increased this preference over sessions, whilst females did not. These data are discussed in relation to sex differences in processing rewards and punishments.

Individual variations in maternal care early in life correlate with later life decision-making and c-fos expression in prefrontal subregions of rats.

Early life adversity affects hypothalamus-pituitary-adrenal axis activity, alters cognitive functioning and in humans is thought to increase the vulnerability to psychopathology--e.g. depression, anxiety and schizophrenia--later in life. Here we investigated whether subtle natural variations among individual rat pups in the amount of maternal care received, i.e. differences in the amount of licking and grooming (LG), correlate with anxiety and prefrontal cortex-dependent behavior in young adulthood. Therefore, we examined the correlation between LG received during the first postnatal week and later behavior in the elevated plus maze and in decision-making processes using a rodent version of the Iowa Gambling Task (rIGT). In our cohort of male and female animals a high degree of LG correlated with less anxiety in the elevated plus maze and more advantageous choices during the last 10 trials of the rIGT. In tissue collected 2 hrs after completion of the task, the correlation between LG and c-fos expression (a marker of neuronal activity) was established in structures important for IGT performance. Negative correlations existed between rIGT performance and c-fos expression in the lateral orbitofrontal cortex, prelimbic cortex, infralimbic cortex and insular cortex. The insular cortex correlations between c-fos expression and decision-making performance depended on LG background; this was also true for the lateral orbitofrontal cortex in female rats. Dendritic complexity of insular or infralimbic pyramidal neurons did not or weakly correlate with LG background. We conclude that natural variations in maternal care received by pups may significantly contribute to later-life decision-making and activity of underlying brain structures.

Rodent versions of the iowa gambling task: opportunities and challenges for the understanding of decision-making.

Impaired decision-making is a core problem in several psychiatric disorders including attention-deficit/hyperactivity disorder, schizophrenia, obsessive-compulsive disorder, mania, drug addiction, eating disorders, and substance abuse as well as in chronic pain. To ensure progress in the understanding of the neuropathophysiology of these disorders, animal models with good construct and predictive validity are indispensable. Many human studies aimed at measuring decision-making capacities use the Iowa gambling task (IGT), a task designed to model everyday life choices through a conflict between immediate gratification and long-term outcomes. Recently, new rodent models based on the same principle have been developed to investigate the neurobiological mechanisms underlying IGT-like decision-making on behavioral, neural, and pharmacological levels. The comparative strengths, as well as the similarities and differences between these paradigms are discussed. The contribution of these models to elucidate the neurobehavioral factors that lead to poor decision-making and to the development of better treatments for psychiatric illness is considered, along with important future directions and potential limitations.

Transient inactivation of the medial prefrontal cortex affects both anxiety and decision-making in male wistar rats.

In both humans and rats high levels of anxiety impair decision-making in the Iowa gambling task (IGT) in male subjects. Expression of the immediate early gene c-fos as marker of neural activity in rat studies indicated a role of the medial prefrontal cortex (prelimbic and infralimbic region; mPFC) in mediating the relationship between anxiety and decision-making. To delineate this relationship further and assess the underlying neurobiology in more detail, we inactivated in the present study the mPFC in male rats using a mixture of the GABA-receptor agonists muscimol and baclofen. Rats were exposed to the elevated plus maze (EPM) to measure effects on anxiety and to the rodent version of the IGT (r-IGT). Inactivation led to increased levels of anxiety on the EPM, while not affecting general activity. The effect in the r-IGT (trials 61-120) was dependent on levels of performance prior to inactivation (trial 41-60): inactivation of the mPFC hampered task performance in rats, which already showed a preference for the advantageous option, but not in rats which were still choosing in a random manner. These data suggest that the mPFC becomes more strongly involved as rats have learned task-contingencies, i.e., choose for the best long-term option. Furthermore they suggest, along with the data of our earlier study, that both anxiety and decision-making in rats are mediated through a neural circuitry including at least the mPFC. The data are discussed in relation to recent data of rodent studies on the neural circuitry underlying decision-making.

Male Wistar rats show individual differences in an animal model of conformity.

Conformity refers to the act of changing one's behaviour to match that of others. Recent studies in humans have shown that individual differences exist in conformity and that these differences are related to differences in neuronal activity. To understand the neuronal mechanisms in more detail, animal tests to assess conformity are needed. Here, we used a test of conformity in rats that has previously been evaluated in female, but not male, rats and assessed the nature of individual differences in conformity. Male Wistar rats were given the opportunity to learn that two diets differed in palatability. They were subsequently exposed to a demonstrator that had consumed the less palatable food. Thereafter, they were exposed to the same diets again. Just like female rats, male rats decreased their preference for the more palatable food after interaction with demonstrator rats that had eaten the less palatable food. Individual differences existed for this shift, which were only weakly related to an interaction between their own initial preference and the amount consumed by the demonstrator rat. The data show that this conformity test in rats is a promising tool to study the neurobiology of conformity.

Repeated rat exposure inhibits the circadian activity patterns of C57BL/6J mice in the home cage.

Exposing male C57BL/6J mice repeatedly, in an unpredictable and uncontrollable fashion to rats, alters their cognitive performance and the neuroendocrine stress response, weeks to months after the rat stress. Continuous observation of the behavioural activity of male C57BL/6J mice in their home cage before (baseline) and after rat exposure could reveal if repeated rat exposure leads to changes in circadian activity patterns, which is a key feature of chronic stress and stress-related disorders in humans. Rat stress (1) decreased exploratory and foraging activity as characterized by increased time spent in the shelter and less time in the open area; (2) reduced sucrose consumption and inhibited the development of sucrose preference, suggesting changes in the reward system and (3) the exploration pattern in a novel environment included more behavioural perseverations, but no change in general locomotor activity. Comparison to baseline activity pattern, i.e., before any intervention, revealed that already the control procedure to rat exposure (spending the same amount of time in another cage) disrupted the organization of behavioural activity patterns, albeit to a different and lesser degree than observed in rat stressed mice. While only the longitudinal design of the study allowed detecting these dynamic patterns of circadian activities, the distinct behavioural changes in foraging and explorative activities support our notion that repeated rat exposure might serve as mouse model of chronic stress.

Effects of genetic background and environmental novelty on wheel running as a rewarding behaviour in mice.

Recent studies suggest running wheel activity to be naturally rewarding and reinforcing; considering the shared neuro-behavioural characteristics with drug-induced reward situations, wheel running behaviour gains interest as a tool to study mechanisms underlying reward-sensitivity. Previously, we showed that wheel running has the potential to disrupt the daily organization of home cage behaviour in female C57BL/6 [de Visser L, van den Bos R, Spruijt BM. Automated home cage observations as a tool to measure the effects of wheel running on cage floor locomotion. Behav Brain Res 2005;160:382-8]. In the present study, we investigated the effects of novelty-induced stress on wheel running and its impact on home cage behaviour in male C57BL/6 and DBA/2 mice. Our aim was to determine whether wheel running may be used as a tool to study both genetic and environmentally induced differences in sensitivity to rewarding behaviour in mice. One group of male mice was placed in an automated home cage observation system for 2 weeks with a wheel integrated in the cage. A second group of mice was allowed to habituate to this cage for 1 week before a running wheel was introduced. Results showed a pronounced sensitising effect of novelty on the level of wheel running in C57Bl/6 mice but not in DBA mice. Overall levels of wheel running were higher in DBA/2 mice. Furthermore, wheel running affected circadian rhythmicity in DBA/2 mice but not in C57BL/6 mice. From these findings we tentatively suggest that wheel running behaviour could serve as a tool to study the interaction between genetic and environmental factors in sensitivity to rewarding behaviour in mice. As it is displayed spontaneously and easy to monitor, wheel running may be well suitable to be included in high-throughput phenotyping assays.

Automated home cage observations as a tool to measure the effects of wheel running on cage floor locomotion.

This paper introduces automated observations in a modular home cage system as a tool to measure the effects of wheel running on the time distribution and daily organization of cage floor locomotor activity in female C57BL/6 mice. Mice (n = 16) were placed in the home cage system for 6 consecutive days. Fifty percent of the subjects had free access to a running wheel that was integrated in the home cage. Overall activity levels in terms of duration of movement were increased by wheel running, while time spent inside a sheltering box was decreased. Wheel running affected the hourly pattern of movement during the animals' active period of the day. Mice without a running wheel, in contrast to mice with a running wheel, showed a clear differentiation between novelty-induced and baseline levels of locomotion as reflected by a decrease after the first day of introduction to the home cage. The results are discussed in the light of the use of running wheels as a tool to measure general activity and as an object for environmental enrichment. Furthermore, the possibilities of using automated home cage observations for e.g. behavioural phenotyping are discussed.