ANCHOR CRC: Results From a Single-Arm, Phase II Study of Encorafenib Plus Binimetinib and Cetuximab in Previously Untreated BRAFV600E-Mutant Metastatic Colorectal Cancer.

PURPOSE: The positive BEACON colorectal cancer (CRC) safety lead-in, evaluating encorafenib + cetuximab + binimetinib in previously treated patients with BRAFV600E-mutated metastatic CRC (mCRC), prompted the design of the phase II ANCHOR CRC study (ClinicalTrails.gov identifier: NCT03693170). ANCHOR CRC aimed to evaluate efficacy, safety, and quality of life with first-line encorafenib + binimetinib + cetuximab in BRAFV600E-mutated mCRC. METHODS: In this multicenter, open-label, single-arm study, patients with BRAFV600E-mutated mCRC received oral encorafenib 300 mg once daily and binimetinib 45 mg twice daily in 28-day cycles, plus intravenous cetuximab 400 mg/m2 once on day 1 of cycle 1, then 250 mg/m2 once weekly for the first seven cycles, and 500 mg/m2 once on Days 1 and 15 from cycle 8 onward. The primary end point was locally assessed confirmed objective response rate (cORR), and secondary end points included centrally assessed cORR, progression-free survival, overall survival (OS), quality of life, and safety and tolerability. RESULTS: Among 95 patients, the locally assessed cORR was 47.4% (95% CI, 37.0 to 57.9) with all partial responses. Since the lower limit of the 95% CI exceeded 30%, the primary end point was met. With a median follow-up duration of 20.1 months, the median progression-free survival on the basis of local assessments was 5.8 months and the median OS was 18.3 months. Treatment was well tolerated, with no unexpected toxicities. Using Patient Global Impression of Changes, substantial improvement in symptoms was consistently reported in ≥ 30% of patients from cycle 3 to cycle 10. CONCLUSION: The ANCHOR CRC study showed that the scientifically driven combination of encorafenib + binimetinib + cetuximab was active in the first-line setting of BRAFV600E-mutated mCRC with a manageable safety profile. Further first-line evaluation is ongoing (ClinicalTrails.gov identifier: NCT04607421).

Olfactory coding in the honeybee lateral horn.

Olfactory systems dynamically encode odor information in the nervous system. Insects constitute a well-established model for the study of the neural processes underlying olfactory perception. In insects, odors are detected by sensory neurons located in the antennae, whose axons project to a primary processing center, the antennal lobe. There, the olfactory message is reshaped and further conveyed to higher-order centers, the mushroom bodies and the lateral horn. Previous work has intensively analyzed the principles of olfactory processing in the antennal lobe and in the mushroom bodies. However, how the lateral horn participates in olfactory coding remains comparatively more enigmatic. We studied odor representation at the input to the lateral horn of the honeybee, a social insect that relies on both floral odors for foraging and pheromones for social communication. Using in vivo calcium imaging, we show consistent neural activity in the honeybee lateral horn upon stimulation with both floral volatiles and social pheromones. Recordings reveal odor-specific maps in this brain region as stimulations with the same odorant elicit more similar spatial activity patterns than stimulations with different odorants. Odor-similarity relationships are mostly conserved between antennal lobe and lateral horn, so that odor maps recorded in the lateral horn allow predicting bees' behavioral responses to floral odorants. In addition, a clear segregation of odorants based on pheromone type is found in both structures. The lateral horn thus contains an odor-specific map with distinct representations for the different bee pheromones, a prerequisite for eliciting specific behaviors.

Appetitive and aversive olfactory learning induce similar generalization rates in the honey bee.

Appetitive and aversive learning drive an animal toward or away from stimuli predicting reinforcement, respectively. The specificity of these memories may vary due to differences in cost­benefit relationships associated with appetitive and aversive contexts. As a consequence, generalization performances may differ after appetitive and aversive training. Here, we determined whether honey bees show different rates of olfactory generalization following appetitive olfactory conditioning of the proboscis extension response, or aversive olfactory conditioning of the sting extension response. In both cases, we performed differential conditioning, which improves discrimination learning between a reinforced odor (CS?) and a non-reinforced odor (CS-) and evaluated generalization to two novel odors whose similarity to the CS? and the CS- was different. We show, given the same level of discriminatory performance, that rates of generalization are similar between the two conditioning protocols and discuss the possible causes for this phenomenon.

Aversive learning overcomes appetitive innate responding in honeybees.

Despite their miniature brain, honeybees have emerged as a powerful model for the study of learning and memory. Yet, they also exhibit innate responses to biologically relevant social signals such as pheromones. Here, we asked whether the bees' developed learning capabilities allow them to overcome hardwired appetitive responses. Can they learn that attractant pheromones, that are not normally associated with a noxious stimulation in nature, predict the punishment of an electric shock? Immobilized honeybees were trained to discriminate two odorants, one that was paired with a shock and another that had no consequences. We measured whether they learned to produce aversive sting extension responses to the punished but not the non-punished odorant. One odorant was a neutral odor without innate value while the other was either an attractive pheromone (geraniol or citral) or an attractive floral odorant (phenylacetaldehyde). In all cases, bees developed a conditioned aversive response to the punished odorant, be it pheromone or not, and efficiently retrieved this information 1 h later. No learning asymmetries between odors were found. Thus, associative aversive learning in bees is strong enough to override preprogrammed responding, thus reflecting an impressive behavioral flexibility.

Visual conditioning of the sting extension reflex in harnessed honeybees.

Visual performances of honeybees have been extensively studied using free-flying individuals trained to choose visual stimuli paired with sucrose reward. By contrast, harnessed bees in the laboratory were not thought to be capable of learning a Pavlovian association between a visual stimulus (CS) and sucrose reward (US). For reasons as yet unknown, harnessed bees only learn visual cues in association with sucrose if their antennae are ablated. However, slow acquisition and low retention performances are obtained in this case. Here, we established a novel visual conditioning protocol, which allows studying visual learning and memory in intact harnessed bees in the laboratory. This protocol consists of conditioning the sting extension reflex (SER) by pairing a visual stimulus (CS+) with an electric shock punishment (US), and a different visual stimulus (CS-) with the absence of shock. Bees with intact antennae learned the discrimination between CS+ and CS- by using chromatic cues, achromatic cues or both. Antennae ablation was not only unnecessary for learning to occur but it even impaired visual SER conditioning because of a concomitant reduction of responsiveness to the electric shock. We thus established the first visual conditioning protocol on harnessed honeybees that does not require injuring the experimental subjects. This novel experimental approach opens new doors for accessing the neural correlates of visual learning and memory in honeybees.

Searching for learning-dependent changes in the antennal lobe: simultaneous recording of neural activity and aversive olfactory learning in honeybees.

Plasticity in the honeybee brain has been studied using the appetitive olfactory conditioning of the proboscis extension reflex, in which a bee learns the association between an odor and a sucrose reward. In this framework, coupling behavioral measurements of proboscis extension and invasive recordings of neural activity has been difficult because proboscis movements usually introduce brain movements that affect physiological preparations. Here we took advantage of a new conditioning protocol, the aversive olfactory conditioning of the sting extension reflex, which does not generate this problem. We achieved the first simultaneous recordings of conditioned sting extension responses and calcium imaging of antennal lobe activity, thus revealing on-line processing of olfactory information during conditioning trials. Based on behavioral output we distinguished learners and non-learners and analyzed possible learning-dependent changes in antennal lobe activity. We did not find differences between glomerular responses to the CS+ and the CS- in learners. Unexpectedly, we found that during conditioning trials non-learners exhibited a progressive decrease in physiological responses to odors, irrespective of their valence. This effect could neither be attributed to a fitness problem nor to abnormal dye bleaching. We discuss the absence of learning-induced changes in the antennal lobe of learners and the decrease in calcium responses found in non-learners. Further studies will have to extend the search for functional plasticity related to aversive learning to other brain areas and to look on a broader range of temporal scales.

Olfactory conditioning of the sting extension reflex in honeybees: Memory dependence on trial number, interstimulus interval, intertrial interval, and protein synthesis.

Harnessed bees learn to associate an odorant with an electric shock so that afterward the odorant alone elicits the sting extension response (SER). We studied the dependency of retention on interstimulus interval (ISI), intertrial interval (ITI), and number of conditioning trials in the framework of olfactory SER conditioning. Forward ISIs (conditioned stimulus [CS] before unconditioned stimulus [US]) supported higher retention than a backward one (US before CS) with an optimum around 3 sec. Spaced trials (ITI 10 min) supported higher retention than massed trials (ITI 1 min) and led to the formation of a late long-term memory (l-LTM) that depended on protein synthesis. Our results reaffirm olfactory SER conditioning as a reliable tool for the study of learning and memory.

Odour aversion after olfactory conditioning of the sting extension reflex in honeybees.

In Pavlovian conditioning, an originally neutral stimulus (conditioned stimulus or CS) gains control over an animal's reflex after its association with a biologically relevant stimulus (unconditioned stimulus or US). As a consequence, a conditioned response is emitted by the animal upon further CS presentations. In such a situation, the subject exhibits a reflex response, so that whether the CS thereby acquires a positive or a negative value for the animal is difficult to assess. In honeybees, Apis mellifera, an odour (CS) can be associated either with sucrose solution (US) in the appetitive conditioning of the proboscis extension reflex (PER), or with an electric shock (US) in the aversive conditioning of the sting extension reflex (SER). The term ;aversive' may not apply to the latter as bees do not suppress SER as a consequence of learning but, on the contrary, start emitting SER to the CS. To determine whether the CS acquires a positive or a negative value in these conditioning forms, we compared the orientation behaviour of freely walking honeybees in an olfactory-cued Y-maze after training them with an odour-sucrose association (PER conditioning) or an odour-shock association (SER conditioning). We show that the same odours can acquire either a positive value when associated to sucrose, or a negative value when associated to an electric shock, as bees respectively approach or avoid the CS in the Y-maze. Importantly, these results clearly establish the aversive nature of SER conditioning in honeybees.

Reappraising social insect behavior through aversive responsiveness and learning.

BACKGROUND: The success of social insects can be in part attributed to their division of labor, which has been explained by a response threshold model. This model posits that individuals differ in their response thresholds to task-associated stimuli, so that individuals with lower thresholds specialize in this task. This model is at odds with findings on honeybee behavior as nectar and pollen foragers exhibit different responsiveness to sucrose, with nectar foragers having higher response thresholds to sucrose concentration. Moreover, it has been suggested that sucrose responsiveness correlates with responsiveness to most if not all other stimuli. If this is the case, explaining task specialization and the origins of division of labor on the basis of differences in response thresholds is difficult. METHODOLOGY: To compare responsiveness to stimuli presenting clear-cut differences in hedonic value and behavioral contexts, we measured appetitive and aversive responsiveness in the same bees in the laboratory. We quantified proboscis extension responses to increasing sucrose concentrations and sting extension responses to electric shocks of increasing voltage. We analyzed the relationship between aversive responsiveness and aversive olfactory conditioning of the sting extension reflex, and determined how this relationship relates to division of labor. PRINCIPAL FINDINGS: Sucrose and shock responsiveness measured in the same bees did not correlate, thus suggesting that they correspond to independent behavioral syndromes, a foraging and a defensive one. Bees which were more responsive to shock learned and memorized better aversive associations. Finally, guards were less responsive than nectar foragers to electric shocks, exhibiting higher tolerance to low voltage shocks. Consequently, foragers, which are more sensitive, were the ones learning and memorizing better in aversive conditioning. CONCLUSIONS: Our results constitute the first integrative study on how aversive responsiveness affects learning, memory and social organization in honeybees. We suggest that parallel behavioral modules (e.g. appetitive, aversive) coexist within each individual bee and determine its tendency to adopt a given task. This conclusion, which is at odds with a simple threshold model, should open new opportunities for exploring the division of labor in social insects.

Aversive learning in honeybees revealed by the olfactory conditioning of the sting extension reflex.

Invertebrates have contributed greatly to our understanding of associative learning because they allow learning protocols to be combined with experimental access to the nervous system. The honeybee Apis mellifera constitutes a standard model for the study of appetitive learning and memory since it was shown, almost a century ago, that bees learn to associate different sensory cues with a reward of sugar solution. However, up to now, no study has explored aversive learning in bees in such a way that simultaneous access to its neural bases is granted. Using odorants paired with electric shocks, we conditioned the sting extension reflex, which is exhibited by harnessed bees when subjected to a noxious stimulation. We show that this response can be conditioned so that bees learn to extend their sting in response to the odorant previously punished. Bees also learn to extend the proboscis to one odorant paired with sugar solution and the sting to a different odorant paired with electric shock, thus showing that they can master both appetitive and aversive associations simultaneously. Responding to the appropriate odorant with the appropriate response is possible because two different biogenic amines, octopamine and dopamine subserve appetitive and aversive reinforcement, respectively. While octopamine has been previously shown to substitute for appetitive reinforcement, we demonstrate that blocking of dopaminergic, but not octopaminergic, receptors suppresses aversive learning. Therefore, aversive learning in honeybees can now be accessed both at the behavioral and neural levels, thus opening new research avenues for understanding basic mechanisms of learning and memory.