Spiral ganglion neurons (SGNs) constitute the sole afferent connection between cochlear hair cells and central auditory nuclei. SGNs die during postnatal developmental pruning, and also following hair cell death, which can be triggered by ototoxic agents such as aminoglycoside antibiotics, including kanamycin. After hair cell loss, animal models show extensive SGN degeneration occurring gradually over a period of weeks to months. Here, we compared spatial and temporal patterns of SGN loss and immune cell involvement in these two cases of cell death in rats. Developmental SGN pruning occurred from postnatal day 5 (P5) to P8 in the basal half of the cochlea, and from P5 to P12 in the apical half. This was accompanied by a transient increase in spiral ganglion macrophages temporally and spatially correlated with SGN death, consistent with a role clearing degenerating neurons. After deafening neonatal rats with kanamycin injections, SGN death became evident at approximately 5.5 weeks of age and persisted throughout the ganglion, with greatest loss in the middle regions; less in the base and apex. Macrophage numbers also increased but neither temporally nor spatially correlated with SGN death. Rather, increased macrophage number and activation began approximately three weeks before SGN death and was highest in the apex. Additionally, T-cells and NK cells appeared in the ganglion concurrently with SGN degeneration. These observations suggest fundamentally different roles for macrophages post-deafening than during developmental pruning and, with prior observations that anti-inflammatory drugs reduce SGN death, support a causal role for immune responses in SGN death post-deafening.

Aminoglycoside antibiotics such as kanamycin induce sensorineural hearing loss by killing hair cells, resulting in secondary degeneration of spiral ganglion neurons (SGNs). Previous studies show that anti-inflammatory agents reduce SGN death, implicating a causal role of the immune response. This is consistent with observations of increased numbers of macrophages and lymphocytes, including T and NK cells, in the spiral ganglion after exposure to aminoglycosides. Here, we directly test the role of T cells and other lymphocytes in SGN degeneration in kanamycin-deafened rats. Homozygous RNU nude rats that lack T cells - but retain NK and B cells- show neurodegeneration similar to rats with a normal T cell complement, indicating that T cells are not necessary for neurodegeneration. Homozygous SRG rats lacking all lymphocytes (i.e., T, B, and NK cell-deficient), exhibit remarkable regional variation in the pattern of spiral ganglion degeneration post-deafening. In the basal half of the ganglion, SGN degeneration is significantly reduced in deafened SRG rats, implying a role for lymphocytes, presumably NK cells of the innate immune system, in SGN death. In the apical half of the deafened ganglion, SGN degeneration is not significantly affected by the lack of all lymphocytes, implying a role for other cellular mechanisms.

The transcription factor ATOH1 is a master regulator of mechanosensory hair-cell (HC) development in the ear. Here, we report that its target gene Casz1 regulates the maturation of outer HCs (OHCs). Genetic deletion of Casz1 during (but not after) cochlear development in the mouse caused: hearing loss; disorganization of mechanosensory stereocilia bundles in OHCs; reduced F-actin density in OHC cuticular plates; progressive OHC loss; and mild morphological alterations in inner HCs. This deletion also altered gene expression, delaying downregulation of genes expressed in immature OHCs, including the actin regulator-encoding gene Coro2a, and accelerating upregulation of genes expressed in mature OHCs. Deleting Coro2a in Casz1 mutant mice restored F-actin density in cuticular plates but increased stereocilia bundle disorganization and hearing thresholds, revealing that CORO2A provides an overall beneficial effect. Our data indicate that CASZ1 regulates transcriptional and morphological maturation of OHCs, and that CASZ1 in maturing HCs is necessary for hearing.

Spiral ganglion neurons (SGNs) transmit auditory information from cochlear hair cells to the brain. SGNs are thus not only important for normal hearing, but also for effective functioning of cochlear implants, which stimulate SGNs when hair cells are missing. SGNs slowly degenerate following aminoglycoside-induced hair cell loss, a process thought to involve an immune response. However, the specific immune response pathways involved remain unknown. We used RNAseq to gain a deeper understanding immune-related and other transcriptomic changes that occur in the rat spiral ganglion after kanamycin-induced deafening. Among the immune and inflammatory genes that were selectively upregulated in deafened spiral ganglia, the complement cascade genes were prominent. We then assessed SGN survival, as well as immune cell numbers and activation, in the spiral ganglia of rats with a CRISPR-Cas9-mediated knockout of complement component 3 (C3). Similar to previous findings in our lab and other deafened rodent models, we observed an increase in macrophage number and increased expression of CD68, a marker of phagocytic activity and cell activation, in macrophages in the deafened ganglia. Moreover, we found an increase in MHCII expression on spiral ganglion macrophages and an increase in lymphocyte number in the deafened ganglia, suggestive of an adaptive immune response. However, C3 knockout did not affect SGN survival or increase in macrophage number/activation, implying that complement activation does not play a role in SGN death after deafening. Together, these data suggest that both innate and adaptive immune responses are activated in the deafened spiral ganglion, with the adaptive response directly contributing to cochlear neurodegeneration.

Introduction Cochlear afferent synapses connecting inner hair cells to spiral ganglion neurons are susceptible to excitotoxic trauma on exposure to loud sound, resulting in a noise-induced cochlear synaptopathy (NICS). Here we assessed the ability of cyclic AMP-dependent protein kinase (PKA) signaling to promote cochlear synapse regeneration, inferred from its ability to promote axon regeneration in axotomized CNS neurons, another system refractory to regeneration.Methods We mimicked NICS in vitro by applying a glutamate receptor agonist, kainic acid (KA) to organotypic cochlear explant cultures and experimentally manipulated cAMP signaling to determine whether PKA could promote synapse regeneration. We then delivered the cAMP phosphodiesterase inhibitor rolipram via implanted subcutaneous minipumps in noise-exposed CBA/CaJ mice to test the hypothesis that cAMP signaling could promote cochlear synapse regeneration in vivo.Results We showed that the application of the cell membrane-permeable cAMP agonist 8-cpt-cAMP or the cAMP phosphodiesterase inhibitor rolipram promotes significant regeneration of synapses in vitro within twelve hours after their destruction by KA. This is independent of neurotrophin-3, which also promotes synapse regeneration. Moreover, of the two independent signaling effectors activated by cAMP - the cAMP Exchange Protein Activated by cAMP and the cAMP-dependent protein kinase - it is the latter that mediates synapse regeneration. Finally, we showed that systemic delivery of rolipram promotes synapse regeneration in vivo following NICS.Discussion In vitro experiments show that cAMP signaling promotes synapse regeneration after excitotoxic destruction of cochlear synapses and does so via PKA signaling. The cAMP phosphodiesterase inhibitor rolipram promotes synapse regeneration in vivo in noise-exposed mice. Systemic administration of rolipram or similar compounds appears to provide a minimally invasive therapeutic approach to reversing synaptopathy post-noise.

Destruction of cochlear hair cells by aminoglycoside antibiotics leads to gradual death of the spiral ganglion neurons (SGNs) that relay auditory information to the brain, potentially limiting the efficacy of cochlear implants. Because the reasons for this cochlear neurodegeneration are unknown, there are no neuroprotective strategies for patients. To investigate this problem, we assessed transcriptomic changes in the rat spiral ganglion following aminoglycoside antibiotic (kanamycin)-induced hair cell destruction. We observed selectively increased expression of immune and inflammatory response genes and increased abundance of activated macrophages in spiral ganglia by postnatal day 32 in kanamycin-deafened rats, preceding significant SGN degeneration. Treatment with the anti-inflammatory medications dexamethasone and ibuprofen diminished long-term SGN degeneration. Ibuprofen and dexamethasone also diminished macrophage activation. Efficacy of ibuprofen treatment was augmented by co-administration of the nicotinamide adenine dinucleotide-stabilizing agent P7C3-A20. Our results support a critical role of neuroinflammation in SGN degeneration after aminoglycoside antibiotic-mediated cochlear hair cell loss, as well as a neuroprotective strategy that could improve cochlear implant efficacy.

Spiral ganglion neurons (SGNs) transmit sensory data from cochlear hair cells to the cochlear nuclei of the brainstem. Loss of these neurons deprives the brain of information on sound, so functioning SGNs are necessary both for normal hearing and for the restoration of hearing with cochlear prostheses. This chapter provides a brief introduction to SGNs and their peripheral and central connections. Then, we summarize three types of SGN pathology and possible methods for their repair, loss of synapses, loss of axons, and loss of neurons entirely. Afferent synapses on inner hair cells can be lost as a consequence of noise exposure or aging, and we discuss experimental approaches to promote synapse regeneration. Aging and noise can also cause death of SGNs, which may occur slowly and involve the degeneration of peripheral axons. We discuss possible approaches to maintaining or regenerating these axons, which may help improve the efficacy of cochlear implants. Finally, we discuss current attempts to use stem cells for the replacement of lost SGNs, along with some of the associated challenges.

Spiral ganglion neurons (SGNs) relay auditory information from cochlear hair cells to the central nervous system. After hair cells are destroyed by aminoglycoside antibiotics, SGNs gradually die. However, the reasons for this cochlear neurodegeneration are unclear. We used microarray gene expression profiling to assess transcriptomic changes in the spiral ganglia of kanamycin-deafened and age-matched control rats and found that many of the genes upregulated after deafening are associated with immune/inflammatory responses. In support of this, we observed increased numbers of macrophages in the spiral ganglion of deafened rats. We also found, via CD68 immunoreactivity, an increase in activated macrophages after deafening. An increase in CD68-associated nuclei was observed by postnatal day 23, a time before significant SGN degeneration is observed. Finally, we show that the immunosuppressive drugs dexamethasone and ibuprofen, as well as the NAD salvage pathway activator P7C3, provide at least some neuroprotection post-deafening. Ibuprofen and dexamethasone also decreased the degree of macrophage activation. These results suggest that activated macrophages specifically, and perhaps a more general neuroinflammatory response, are actively contributing to SGN degeneration after hair cell loss. ### Competing Interest Statement The authors have declared no competing interest.

Loud noise damages the auditory sensory cells—the cochlear hair cells—which generally results in an elevated auditory threshold detectable on conventional hearing tests. However, loss of hair cells is not the only consequence of noise. Hair cells communicate auditory information to the brain via the spiral ganglion neurons, whose axons form the auditory nerve. Noise also damages the synaptic connections between hair cells and spiral ganglion neurons, disrupting that vital communication and reducing activity in the auditory nerve.1 This damage to synapses, termed synaptopathy, is, in some respects, more insidious than the loss of hair cells. Cochlear synaptopathy can be caused by noise levels lower than those that damage hair cells2, and is therefore likely to be more common. Postmortem observations of human cochleae show intact hair cells with diminished numbers of synapses.3 Because the hair cells remain, auditory thresholds are not significantly elevated and simply measuring thresholds will not reveal any impairment. Consequently, hearing impairment caused by synaptopathy alone is often referred to as hidden hearing loss. The damage is detectable using auditory tests other than measuring threshold. For example, reduced activity in the auditory nerve is detectable by the measure of the auditory brainstem response.4 Hidden hearing loss has been conjectured to be a possible cause of tinnitus and compromised speech comprehension in noisy environments.2iStock/Rasi Bhadramani, hearing loss, research, audiology.NOISE-INDUCED COCHLEAR SYNAPTOPATHY Lost hair cells do not regenerate in mammals nor do lost cochlear synapses, although attempts to find means to promote such regeneration are underway in many laboratories. Our laboratory, however, is investigating the prevention of noise-induced cochlear synaptopathy (NICS). To this end, we have investigated the cause of synaptopathy by focusing on the physiology of the cochlear synapse. These synapses use glutamate as the neurotransmitter.5,6 The hair cell, the presynaptic side of the synapse, releases glutamate in response to sound. Glutamate binds to receptors on the spiral ganglion neuron at the postsynaptic side of the synapse.7,8 The glutamate receptors then allow an ionic current to flow into the postsynaptic structure, thereby initiating electrical activity in the auditory nerve. Louder sound results in larger amounts of glutamate released from the hair cell, which increases activity in the auditory nerve to inform the brain about the loudness of the sound. With moderate sound levels, this process of normal synaptic transmission functions without causing any damage. However, very loud sounds can cause excess glutamate release from the hair cell, resulting in greatly increased postsynaptic inward current, including the increased inward flow of calcium ions (Ca2+). Studies of glutamatergic synapses in the brain had already shown that such inward flow of Ca2+ can result in damage to the postsynaptic cell, a phenomenon termed excitotoxicity.9 The possibility that NICS is due to excitotoxicity was investigated by Pujol and colleagues, who showed that direct infusion of glutamate agonists into the cochlea of guinea pigs caused excitotoxic destruction of cochlear synapses resembling damage caused by noise.10,11 They further showed that pharmacological blockade of glutamate receptors prevented NICS.12 These studies established that the proximate cause of NICS is excitotoxicity due to the excessive release of glutamate from hair cells at cochlear synapses. However, blockade of all glutamate receptors is not an optimal means to protect cochlear synapses from noise because such blockade would entirely prevent synaptic transmission and hearing. A more precisely targeted approach is needed, recognizing that glutamate receptors are diverse and not all types of glutamate receptors may be responsible for the damage. An important clue is that excitotoxicity is generally associated with Ca2+ influx and not all glutamate receptors are permeable to Ca2+. In the brain, excitotoxic Ca2+ influx is mainly via NMDA-type glutamate receptors, but this does not appear to be the case in the cochlea. Glutamate agonists that do not activate NMDA receptors can account for excitotoxicity in the cochlea.10 Rather, these studies implicated AMPA-type glutamate receptors, often referred to as AMPA receptors, which are tetramers made up of combinations of four different subunits, GluA1, GluA2, GluA3, and GluA4, encoded by four different genes, Gria1, Gria2, Gria3, and Gria4, respectively. The subunit composition of individual AMPA receptors depends on which genes are expressed in a cell. For example, in the brain, GluA1 is often a predominant subunit and AMPA receptors will typically have at least one of the subunits being GluA1. In the cochlea, spiral ganglion neurons express little, if any, GluA1 and AMPA receptors at the hair cell synapse that are composed of GluA2, GluA3, and GluA4 subunits.13 What is most relevant to the issue of excitotoxicity is GluA2. AMPA receptors with one or more GluA2 subunits in the tetramer are impermeable to calcium. AMPA receptors lacking GluA2 subunits are calcium permeable and are therefore capable of contributing to excitotoxicity.14,15 STUDY HIGHLIGHTS Our laboratory took advantage of a paradigm that had been previously developed by Kujawa, Liberman, and their colleagues16 to calibrate noise exposure to mice at a level that caused synaptopathy but that did not damage hair cells. By confining the damage to the postsynaptic elements, it was possible to test compounds that would be specifically protective against synaptopathy. We used IEM-1460, a compound that selectively blocks GluA2-lacking AMPA receptors,17,18 to ask whether these could be the culprits in NICS. We found that when introduced into the cochlea of mice prior to noise and present throughout the noise exposure, IEM-1460 effectively prevented synaptopathy, as demonstrated by the counts of cochlear synapses and the auditory nerve activity in the auditory brainstem response (ABR).19 This result was somewhat unexpected because we know that GluA2 is present at cochlear synapses. This was resolved by observations made by our colleague Mark Rutherford, PhD, who showed that on a nanoscale, the postsynaptic side of the synapse contains patches of AMPA receptors that are deficient in GluA2 and patches in which GluA2 is more abundant.19 Thus, despite the presence of GluA2 at the synapse, GluA2-lacking AMPA receptors can be responsible for excitotoxic trauma to the postsynaptic element. Blocking these receptors with IEM-1460 prevents excitotoxic trauma and so prevents NICS. The mice appeared to have normal hearing as assessed by ABR measures even while the cochlea was being infused with IEM-1460.19 Presumably, this is because the synapse does have AMPA receptors containing GluA2. These are not blocked by IEM-1460, so they can maintain synaptic transmission even while the GluA2-lacking AMPA receptors are blocked, but the GluA2-containing AMPA receptors do not contribute to excitotoxicity. Thus, the selective blocker of GluA2-lacking AMPA receptors can prevent NICS while not interfering with normal hearing, something not achievable with less selective compounds that block all glutamate receptors or all AMPA-type glutamate receptors. In principle, such compounds could be used to protect individuals against NICS in loud work environments (e.g., the military) without preventing their ability to hear orders, warnings, or other communications. However, this “chemical earmuff” strategy has only been assessed in mice, and it is yet to be established whether such an approach would be safe and effective in humans.

Exposure to loud sound damages the postsynaptic terminals of spiral ganglion neurons (SGNs) on cochlear inner hair cells (IHCs), resulting in loss of synapses, a process termed synaptopathy. Glutamatergic neurotransmission via α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA)-type receptors is required for synaptopathy, and here we identify a possible involvement of GluA2-lacking Ca2+-permeable AMPA receptors (CP-AMPARs) using IEM-1460, which has been shown to block GluA2-lacking AMPARs. In CBA/CaJ mice, a 2-h exposure to 100-dB sound pressure level octave band (8 to 16 kHz) noise results in no permanent threshold shift but does cause significant synaptopathy and a reduction in auditory brainstem response (ABR) wave-I amplitude. Chronic intracochlear perfusion of IEM-1460 in artificial perilymph (AP) into adult CBA/CaJ mice prevented the decrease in ABR wave-I amplitude and the synaptopathy relative to intracochlear perfusion of AP alone. Interestingly, IEM-1460 itself did not affect the ABR threshold, presumably because GluA2-containing AMPARs can sustain sufficient synaptic transmission to evoke low-threshold responses during blockade of GluA2-lacking AMPARs. On individual postsynaptic densities, we observed GluA2-lacking nanodomains alongside regions with robust GluA2 expression, consistent with the idea that individual synapses have both CP-AMPARs and Ca2+-impermeable AMPARs. SGNs innervating the same IHC differ in their relative vulnerability to noise. We found local heterogeneity among synapses in the relative abundance of GluA2 subunits that may underlie such differences in vulnerability. We propose a role for GluA2-lacking CP-AMPARs in noise-induced cochlear synaptopathy whereby differences among synapses account for differences in excitotoxic susceptibility. These data suggest a means of maintaining normal hearing thresholds while protecting against noise-induced synaptopathy, via selective blockade of CP-AMPARs.

Together with the recent CLIC detector model CLICdet a new software suite was introduced for the simulation and reconstruction of events in this detector. This note gives a brief introduction to CLICdet and describes the CLIC experimental conditions at 380 GeV and 3 TeV, including beam-induced backgrounds. The simulation and reconstruction tools are introduced, and the physics performance obtained is described in terms of single particles, particles in jets, jet energy resolution and flavour tagging. The performance of the very forward electromagnetic calorimeters is also discussed.

The Compact Linear Collider (CLIC) is an option for a future electron-positron collider operating at centre-of-mass energies from a few hundred GeV up to 3 TeV. Details will be presented on two recent physics benchmark analyses of electroweak measurements at CLIC based on full detector simulations and assuming centre-of-mass energies of 1.4 and 3 TeV. Vector boson scattering gives insight into the mechanism of electroweak symmetry breaking. The processes $\mathrm{e^{+}e^{-}}\rightarrow\mathrm{WW}\nu\nu$ and $\mathrm{e^{+}e^{-}}\rightarrow\mathrm{ZZ}\nu\nu$ were studied using fully hadronic events which provide the full kinematic information on the final-state bosons. The expected precisions on anomalous gauge couplings are extracted. The process $\mathrm{e^{+}e^{-}}\rightarrow\gamma\gamma$ allows to search for deviations from QED. The expected sensitivities to a finite electron size and other scenarios are discussed.

CaBPs are a family of Ca2+ binding proteins related to calmodulin. Two CaBP family members, CaBP1 and CaBP2, are highly expressed in the cochlea. Here, we investigated the significance of CaBP1 and CaBP2 for hearing in mice lacking expression of these proteins (CaBP1 KO and CaBP2 KO) using auditory brain responses (ABRs) and distortion product otoacoustic emissions (DPOAEs). In CaBP1 KO mice, ABR wave I was larger in amplitude, and shorter in latency and faster in decay, suggestive of enhanced synchrony of auditory nerve fibers. This interpretation was supported by the greater excitability of CaBP1 KO than WT neurons in whole-cell patch clamp recordings of spiral ganglion neurons in culture, and normal presynaptic function of CaBP1 KO IHCs. DPOAEs and ABR thresholds were normal in 4-week old CaBP1 KO mice, but elevated ABR thresholds became evident at 32 kHz at 9 weeks, and at 8 and 16 kHz by 6 months of age. In contrast, CaBP2 KO mice exhibited significant ABR threshold elevations at 4 weeks of age that became more severe in the mid-frequency range by 9 weeks. Though normal at 4 weeks, DPOAEs in CaBP2 KO mice were significantly reduced in the mid-frequency range by 9 weeks. Our results reveal requirements for CaBP1 and CaBP2 in the peripheral auditory system and highlight the diverse modes by which CaBPs influence sensory processing.

The particle flow approach to calorimetry benefits from highly granular calorimeters and sophisticated software algorithms in order to reconstruct and identify individual particles in complex event topologies. The high spatial granularity, together with analogue energy information, can be further exploited in software compensation. In this approach, the local energy density is used to discriminate electromagnetic and purely hadronic sub-showers within hadron showers in the detector to improve the energy resolution for single particles by correcting for the intrinsic non-compensation of the calorimeter system. This improvement in the single particle energy resolution also results in a better overall jet energy resolution by improving the energy measurement of identified neutral hadrons and improvements in the pattern recognition stage by a more accurate matching of calorimeter energies to tracker measurements. This paper describes the software compensation technique and its implementation in particle flow reconstruction with the Pandora Particle Flow Algorithm (PandoraPFA). The impact of software compensation on the choice of optimal transverse granularity for the analogue hadronic calorimeter option of the International Large Detector (ILD) concept is also discussed.

Honor Fell first succeeded in culture of the cochlea in 1928. Throughout most of the time since then, the principal use of cochlear or spiral ganglion cultures has been to facilitate the study of cochlear development: cell differentiation, structural and physiological maturation, and innervation of the sensory cells. More recently, use of spiral ganglion cultures has been extended to additional issues. One of these is neuronal survival in response to neurotrophic factors and electrical activity. The goal of such studies is to prevent degeneration or death of spiral ganglion neurons (SGNs) in the hearing impaired and so improve efficacy of cochlear implants. A second topic is degeneration or death of SGNs following direct trauma, particularly noise-induced excitotoxic trauma. The goal of these studies is to identify means of protecting the SGNs and their synapses on hair cells following trauma and to promote regeneration. The success of these studies has been due to clever exploitation of several technical innovations. Culture conditions have been improved by the use of serum and specialized culture media and the use of more physiological substrate materials. Electrophysiological methods have been applied. Microscopy has advanced from conventional optical methods to the use of electron, conventional fluorescence, and confocal microscopy, greatly improving observations of fixed or live cochlear cultures. Highly specialized substrates have allowed study of SGN axon guidance by chemical or physical cues. There is a greatly expanded repertoire of small molecule pharmacological agents, peptide growth and trophic factors targeting surface molecules and receptors, and cell membrane-permeable reagents for modulating intracellular signaling and regulation. Further expanding the range of experimental manipulations has been the introduction of molecular and genetic techniques, including gene transfer by transfection or viral transduction and culture of cells or organs from transgenic mice. Here we provide a historical overview of the study of the spiral ganglion in vitro noting key experiments exemplifying the application of increasingly sophisticated experimental and observational techniques to the research goals. We hope that readers will obtain, from this overview, ideas for experimental approaches applicable to their own research questions.

legend N2D N1D 2LPEG N2D vs. 2LPEG N1D vs. 2LPEG EFFICACY Primary analysis set, n1⁄4 275 Primary analysis set, n1⁄4 275 Primary analysis set, n1⁄4 272 Primary endpoint: Patients with successful overall bowel cleansing efficacy (HCS) [n] 253 (92.0%) 245 (89.1%) 238 (87.5%) -4.00%* [0.055] -6.91%* [0.328] Supportive secondary endpoint: Patients with successful overall bowel cleansing efficacy (BBPS) [n] 249 (90.5%) 243 (88.4%) 232 (85.3%) n.a. n.a. Primary endpoint: Excellent plus Good cleansing rate in colon ascendens (primary analysis set) [n] 87 (31.6%) 93 (33.8%) 41 (15.1%) 8.11%* [50.001] 10.32%* [50.001] Key secondary endpoint: Adenoma detection rate, colon ascendens 11.6% 11.6% 8.1% -4.80%; 12.00%** [0.106] -4.80%; 12.00%** [0.106] Key secondary endpoint: Adenoma detection rate, overall colon 26.6% 27.6% 26.8% -8.47%; 8.02%** [0.569] -7.65%; 9.11%** [0.455] Key secondary endpoint: Polyp detection rate, colon ascendens 23.3% 18.6% 16.2% -1.41%; 15.47%** [0.024] -6.12%; 10.82%** [0.268] Key secondary endpoint: Polyp detection rate, overall colon 44.0% 45.1% 44.5% -8.85%; 8.00%** [0.579] –7.78%; 9.09%** [0.478] Compliance rates (min 75% of both doses taken) [n] 235 (85.5%) 233 (84.7%) 245 (90.1%) n.a. n.a. SAFETY Safety set, n1⁄4 262 Safety set, n1⁄4 269 Safety set, n1⁄4 263 All treatment-emergent adverse events [n] 77 89 53 n.a. n.a. Patients with any related treatment-emergent adverse event [n] 30 (11.5%) 40 (14.9%) 20 (7.6%) n.a. n.a. *1⁄4 97.5% 1-sided CI; **1⁄4 95% 2-sided CI; n.a.1⁄4 not applicable. United European Gastroenterology Journal 4(5S) A219

Background Nilotinib inhibits the tyrosine kinase activity of ABL1/BCR-ABL1 and KIT, platelet-derived growth factor receptors (PDGFRs), and the discoidin domain receptor. Gain-of-function mutations in KIT or PDGFR alpha are key drivers in most gastrointestinal stromal tumours (GISTs). This trial was designed to test the efficacy and safety of nilotinib versus imatinib as first-line therapy for patients with advanced GISTs.Methods In this randomised, open-label, multicentre, phase 3 trial (ENESTg1), participants from academic centres were aged 18 years or older and had previously untreated, histologically confirmed, metastatic or unresectable GISTs. Patients were stratified by previous adjuvant therapy and randomly assigned (1: 1) via a randomisation list to receive oral imatinib 400 mg once daily or oral nilotinib 400 mg twice daily. The primary endpoint was centrally reviewed progression-free survival. Efficacy endpoints were assessed by intention-to-treat. This trial is registered with ClinicalTrials.gov, number NCT00785785.Findings Because the futility boundary was crossed at a preplanned interim analysis, trial accrual terminated in April, 2011. Between March 16, 2009, and April 21, 2011, 647 patients were enrolled; of whom 324 were allocated nilotinib and 320 were allocated imatinib. At final analysis of the core study (data cutoff, October, 2012), 2-year progression-free survival was higher in the imatinib group (59.2% [95% CI 50.9-66.5]) than in the nilotinib group (51.6% [43.0-59.5]; hazard ratio 1.47 [95% CI 1.10-1.95]). In the imatinib group, the most common grade 3-4 adverse events were hypophosphataemia (19 [6%]), anaemia (17 [5%]), abdominal pain (13; 4%), and elevated lipase level (15; 5%), and in the nilotinib group were anaemia (18; 6%), elevated lipase level (15; 5%), elevated alanine aminotransferase concentration (12; 4%), and abdominal pain (11; 3%). The most common serious adverse event in both groups was abdominal pain (11 [4%] in the imatinib group, 14 [4%] in the nilotinib group).Interpretation Nilotinib cannot be recommended for broad use to treat first-line GIST. However, future studies might identify patient subsets for whom first-line nilotinib could be of clinical benefit.

In the ENESTg1 study, Blay and colleagues 1 Blay JY Shen L Kang YK et al. Nilotinib versus imatinib as first-line therapy for patients with unresectable or metastatic gastrointestinal stromal tumours (ENESTg1): a randomised phase 3 trial. Lancet Oncol. 2015; 16: 550-560 Summary Full Text Full Text PDF PubMed Scopus (75) Google Scholar report large differences between the activities of imatinib and nilotinib as first-line treatment for patients with advanced gastrointestinal stromal tumours (GIST). When analysed by KIT mutation status, imatinib and nilotinib seemed to have similar activity in patients with KIT exon 11 mutations, but imatinib was more active than nilotinib in patients with tumours harbouring a KIT exon 9 mutation. However, as mentioned by Blay and colleagues, preclinical data are not in line with such clinical findings, since imatinib and nilotinib show similar in-vitro activity against tumour cells harbouring KIT exon 9 mutations. We believe that, beyond tumour molecular characteristics, other patient-related factors could have affected the pharmacokinetics of nilotinib but not those of imatinib, thereby resulting in different anti-tumour activity. Nilotinib versus imatinib as first-line therapy for patients with unresectable or metastatic gastrointestinal stromal tumours (ENESTg1): a randomised phase 3 trialNilotinib cannot be recommended for broad use to treat first-line GIST. However, future studies might identify patient subsets for whom first-line nilotinib could be of clinical benefit. Full-Text PDF Nilotinib versus imatinib for GIST – Authors' replyWe acknowledge the interesting points raised by Paul Gougis and colleagues with respect to the differential effects of patient-related and treatment-related factors on the pharmacokinetics of nilotinib compared with imatinib, and their potential effects on anti-tumour activity observed in the ENESTg trial.1 Our trial1 was not specifically designed to examine these associations and no specific analyses of these issues were pre-planned. However, analysis of the dataset and published literature regarding previous gastrectomy and use of proton pump inhibitors suggests that a large effect on overall efficacy results is unlikely. Full-Text PDF