Molecular profiling of metastatic breast cancer and target-based therapeutic matching in an Asian tertiary phase I oncology unit.

Introduction: Molecular profiling of metastatic breast cancer (MBC) through the widespread use of next-generation sequencing (NGS) has highlighted actionable mutations and driven trials of targeted therapy matched to tumour molecular profiles, with improved outcomes reported using such an approach. Here, we review NGS results and treatment outcomes for a cohort of Asian MBC patients in the phase I unit of a tertiary centre. Methods: Patients with MBC referred to a phase I unit underwent NGS via Ion AmpliSeq Cancer Hotspot v2 (ACH v2, 2014-2017) prior to institutional change to FoundationOne CDx (FM1; 2017-2022). Patients were counselled on findings and enrolled on matched therapeutic trials, where available. Outcomes for all subsequent treatment events were recorded to data cut-off on January 31, 2022. Results: A total of 215 patients were enrolled with successful NGS in 158 patients. The PI3K/AKT/PTEN pathway was the most altered with one or more of the pathway member genes PIK3/AKT/PTEN affected in 62% (98/158) patients and 43% of tumours harbouring a PIK3CA alteration. Tumour mutational burden (TMB) was reported in 96/109 FM1 sequenced patients, with a mean TMB of 5.04 mt/Mb and 13% (12/96) with TMB ≥ 10 mt/Mb. Treatment outcomes were evaluable in 105/158 patients, with a pooled total of 216 treatment events recorded. Matched treatment was administered in 47/216 (22%) events and associated with prolonged median progression-free survival (PFS) of 21.0 weeks [95% confidence interval (CI) 11.7, 26.0 weeks] versus 12.1 weeks (95% CI 10.0, 15.4 weeks) in unmatched, with hazard ratio (HR) for progression or death of 0.63 (95% CI 0.41, 0.97; p = 0.034). In the subgroup of PIK3/AKT/PTEN-altered MBC, the HR for progression or death was 0.57 (95% CI 0.35, 0.92; p = 0.02), favouring matched treatment. Per-patient overall survival (OS) analysis (n = 105) showed improved survival for patients receiving matched treatment versus unmatched, with median OS (mOS) of 30.1 versus 11.8 months, HR = 0.45 (95% CI 0.24, 0.84; p = 0.013). Objective response rate (ORR) in the overall population was similar in matched and unmatched treatment events (23.7% versus 17.2%, odds ratio of response 1.14 95% CI 0.50, 2.62; p = 0.75). Conclusions: Broad-panel NGS in MBC is feasible, allowing therapeutic matching, which was associated with improvements in PFS and OS.

Excitatory Spinal Lhx9-Derived Interneurons Modulate Locomotor Frequency in Mice.

Locomotion allows us to move and interact with our surroundings. Spinal networks that control locomotion produce rhythm and left-right and flexor-extensor coordination. Several glutamatergic populations, Shox2 non-V2a, Hb9-derived interneurons, and, recently, spinocerebellar neurons have been proposed to be involved in the mouse rhythm generating networks. These cells make up only a smaller fraction of the excitatory cells in the ventral spinal cord. Here, we set out to identify additional populations of excitatory spinal neurons that may be involved in rhythm generation or other functions in the locomotor network. We use RNA sequencing from glutamatergic, non-glutamatergic, and Shox2 cells in the neonatal mice from both sexes followed by differential gene expression analyses. These analyses identified transcription factors that are highly expressed by glutamatergic spinal neurons and differentially expressed between Shox2 neurons and glutamatergic neurons. From this latter category, we identified the Lhx9-derived neurons as having a restricted spinal expression pattern with no Shox2 neuron overlap. They are purely glutamatergic and ipsilaterally projecting. Ablation of the glutamatergic transmission or acute inactivation of the neuronal activity of Lhx9-derived neurons leads to a decrease in the frequency of locomotor-like activity without change in coordination pattern. Optogenetic activation of Lhx9-derived neurons promotes locomotor-like activity and modulates the frequency of the locomotor activity. Calcium activities of Lhx9-derived neurons show strong left-right out-of-phase rhythmicity during locomotor-like activity. Our study identifies a distinct population of spinal excitatory neurons that regulates the frequency of locomotor output with a suggested role in rhythm-generation in the mouse alongside other spinal populations.

Management of Oncogene Driven Locally Advanced Unresectable Non-small Cell Lung Cancer.

INTRODUCTION: The current standard of care of locally advanced non-small cell lung cancer (LA-NSCLC) is concurrent chemoradiation, followed by consolidation durvalumab. However, there is evidence that the efficacy of chemoradiation and also immunotherapy in many oncogene-positive LA-NSCLC are attenuated, and dependent on the subgroup. AREAS COVERED: We will firstly review the outcomes of standard-of-care therapy in oncogene-driven LA-NSCLC. We looked at various oncogene driven subgroups and the tumor microenvironment that may explain differential response. Finally, we review the role of targeted therapy in the treatment of LA-NSCLC. EXPERT OPINION: Each oncogene-positive subgroup should be treated as its own entity, and continued efforts should be undertaken to incorporate targeted therapy, which is likely to yield superior survival outcomes if trial design can be optimized and toxicities can be managed.

Developmental program-independent secretory granule degradation in larval salivary gland cells of Drosophila.

Both constitutive and regulated secretion require cell organelles that are able to store and release the secretory cargo. During development, the larval salivary gland of Drosophila initially produces high amount of glue-containing small immature secretory granules, which then fuse with each other and reach their normal 3-3.5 µm in size. Following the burst of secretion, obsolete glue granules directly fuse with late endosomes or lysosomes by a process called crinophagy, which leads to fast degradation and recycling of the secretory cargo. However, hindering of endosome-to-TGN retrograde transport in these cells causes abnormally small glue granules which are not able to fuse with each other. Here, we show that loss of function of the SNARE genes Syntaxin 16 (Syx16) and Synaptobrevin (Syb), the small GTPase Rab6 and the GARP tethering complex members Vps53 and Scattered (Vps54) all involved in retrograde transport cause intense early degradation of immature glue granules via crinophagy independently of the developmental program. Moreover, silencing of these genes also provokes secretory failure and accelerated crinophagy during larval development. Our results provide a better understanding of the relations among secretion, secretory granule maturation and degradation and paves the way for further investigation of these connections in other metazoans.

Ecdysone receptor isoform specific regulation of secretory granule acidification in the larval Drosophila salivary gland.

Bulk production and release of glue containing secretory granules takes place in the larval salivary gland during Drosophila development in order to attach the metamorphosing animal to a dry surface. These granules undergo a maturation process to prepare glue for exocytosis, which includes homotypic fusions to increase the size of granules, vesicle acidification and ion uptake. The steroid hormone 20-hydroxyecdysone is known to be required for the first and last steps of this process: glue synthesis and secretion, respectively. Here we show that the B1 isoform of Ecdysone receptor (EcR), together with its binding partner Ultraspiracle, are also necessary for the maturation of glue granules by promoting their acidification via regulation of Vha55 expression, which encodes an essential subunit of the V-ATPase proton pump. This is antagonized by the EcR-A isoform, overexpression of which decreases EcR-B1 and Vha55 expression and glue granule acidification. Our data shed light on a previously unknown, ecdysone receptor isoform-specific regulation of glue granule maturation.

Extracellular Vesicle-Mediated Metastasis Suppressors NME1 and NME2 Modify Lipid Metabolism in Fibroblasts.

Nowadays, extracellular vesicles (EVs) raise a great interest as they are implicated in intercellular communication between cancer and stromal cells. Our aim was to understand how vesicular NME1 and NME2 released by breast cancer cells influence the tumour microenvironment. As a model, we used human invasive breast carcinoma cells overexpressing NME1 or NME2, and first analysed in detail the presence of both isoforms in EV subtypes by capillary Western immunoassay (WES) and immunoelectron microscopy. Data obtained by both methods showed that NME1 was present in medium-sized EVs or microvesicles, whereas NME2 was abundant in both microvesicles and small-sized EVs or exosomes. Next, human skin-derived fibroblasts were treated with NME1 or NME2 containing EVs, and subsequently mRNA expression changes in fibroblasts were examined. RNAseq results showed that the expression of fatty acid and cholesterol metabolism-related genes was decreased significantly in response to NME1 or NME2 containing EV treatment. We found that FASN (fatty acid synthase) and ACSS2 (acyl-coenzyme A synthetase short-chain family member 2), related to fatty acid synthesis and oxidation, were underexpressed in NME1/2-EV-treated fibroblasts. Our data show an emerging link between NME-containing EVs and regulation of tumour metabolism.

Different Metabolism and Toxicity of TRANS Fatty Acids, Elaidate and Vaccenate Compared to Cis-Oleate in HepG2 Cells.

Trans fatty acids (TFAs) are not synthesized in the human body but are generally ingested in substantial amounts. The widespread view that TFAs, particularly those of industrial origin, are unhealthy and contribute to obesity, cardiovascular diseases and diabetes is based mostly on in vivo studies, and the underlying molecular mechanisms remain to be elucidated. Here, we used a hepatoma model of palmitate-induced lipotoxicity to compare the metabolism and effects of the representative industrial and ruminant TFAs, elaidate and vaccenate, respectively, with those of cis-oleate. Cellular FAs, triacylglycerols, diacylglycerols and ceramides were quantitated using chromatography, markers of stress and apoptosis were assessed at mRNA and protein levels, ultrastructural changes were examined by electron microscopy and viability was evaluated by MTT assay. While TFAs were just slightly more damaging than oleate when applied alone, they were remarkably less protective against palmitate toxicity in cotreatments. These differences correlated with their diverse incorporation into the accumulating diacylglycerols and ceramides. Our results provide in vitro evidence for the unfavorable metabolic features and potent stress-inducing character of TFAs in comparison with oleate. These findings strengthen the reasoning against dietary trans fat intake, and they can also help us better understand the molecular mechanisms of lipotoxicity.

Case scenario: Management of major depressive disorder in primary care based on the updated Malaysian clinical practice guidelines.

Major depressive disorder (MDD) is a common but complex illness that is frequently presented in the primary care setting. Managing this disorder in primary care can be difficult, and many patients are underdiagnosed and/or undertreated. The Malaysian Clinical Practice Guidelines (CPG) on the Management of Major Depressive Disorder (MDD) (2nd ed.), published in 2019, covers screening, diagnosis, treatment and referral (which frequently pose a challenge in the primary care setting) while minimising variation in clinical practice.

Locomotor deficits in a mouse model of ALS are paralleled by loss of V1-interneuron connections onto fast motor neurons.

ALS is characterized by progressive inability to execute movements. Motor neurons innervating fast-twitch muscle-fibers preferentially degenerate. The reason for this differential vulnerability and its consequences on motor output is not known. Here, we uncover that fast motor neurons receive stronger inhibitory synaptic inputs than slow motor neurons, and disease progression in the SOD1G93A mouse model leads to specific loss of inhibitory synapses onto fast motor neurons. Inhibitory V1 interneurons show similar innervation pattern and loss of synapses. Moreover, from postnatal day 63, there is a loss of V1 interneurons in the SOD1G93A mouse. The V1 interneuron degeneration appears before motor neuron death and is paralleled by the development of a specific locomotor deficit affecting speed and limb coordination. This distinct ALS-induced locomotor deficit is phenocopied in wild-type mice but not in SOD1G93A mice after appearing of the locomotor phenotype when V1 spinal interneurons are silenced. Our study identifies a potential source of non-autonomous motor neuronal vulnerability in ALS and links ALS-induced changes in locomotor phenotype to inhibitory V1-interneurons.

The Role of Deubiquitinating Enzymes in the Various Forms of Autophagy.

Deubiquitinating enzymes (DUBs) have an essential role in several cell biological processes via removing the various ubiquitin patterns as posttranslational modification forms from the target proteins. These enzymes also contribute to the normal cytoplasmic ubiquitin pool during the recycling of this molecule. Autophagy, a summary name of the lysosome dependent self-degradative processes, is necessary for maintaining normal cellular homeostatic equilibrium. Numerous forms of autophagy are known depending on how the cellular self-material is delivered into the lysosomal lumen. In this review we focus on the colorful role of DUBs in autophagic processes and discuss the mechanistic contribution of these molecules to normal cellular homeostasis via the possible regulation forms of autophagic mechanisms.

Early delivery and prolonged treatment with nimodipine prevents the development of spasticity after spinal cord injury in mice.

Spasticity, one of the most frequent comorbidities of spinal cord injury (SCI), disrupts motor recovery and quality of life. Despite major progress in neurorehabilitative and pharmacological approaches, therapeutic strategies for treating spasticity are lacking. Here, we show in a mouse model of chronic SCI that treatment with nimodipine-an L-type calcium channel blocker already approved from the European Medicine Agency and from the U.S. Food and Drug Administration-starting in the acute phase of SCI completely prevents the development of spasticity measured as increased muscle tone and spontaneous spasms. The aberrant muscle activities associated with spasticity remain inhibited even after termination of the treatment. Constitutive and conditional silencing of the L-type calcium channel CaV1.3 in neuronal subtypes demonstrated that this channel mediated the preventive effect of nimodipine on spasticity after SCI. This study identifies a treatment protocol and suggests that targeting CaV1.3 could prevent spasticity after SCI.

Comparing the effects of uncoated nanostructured surfaces on primary neurons and astrocytes.

The long-term application of central nervous system implants is currently limited by the negative response of the brain tissue, affecting both the performance of the device and the survival of nearby cells. Topographical modification of implant surfaces mimicking the structure and dimensions of the extracellular matrix may provide a solution to this negative tissue response and has been shown to affect the attachment and behavior of both neurons and astrocytes. In our study, commonly used neural implant materials, silicon, and platinum were tested with or without nanoscale surface modifications. No biological coatings were used in order to only examine the effect of the nanostructuring. We seeded primary mouse astrocytes and hippocampal neurons onto four different surfaces: flat polysilicon, nanostructured polysilicon, and platinum-coated versions of these surfaces. Fluorescent wide-field, confocal, and scanning electron microscopy were used to characterize the attachment, spreading and proliferation of these cell types. In case of astrocytes, we found that both cell number and average cell spreading was significantly larger on platinum, compared to silicon surfaces, while silicon surfaces impeded glial proliferation. Nanostructuring did not have a significant effect on either parameter in astrocytes but influenced the orientation of actin filaments and glial fibrillary acidic protein fibers. Neuronal soma attachment was impaired on metal surfaces while nanostructuring seemed to influence neuronal growth cone morphology, regardless of surface material. Taken together, the type of metals tested had a profound influence on cellular responses, which was only slightly modified by nanopatterning.

Decreased Nuclear Ascorbate Accumulation Accompanied with Altered Genomic Methylation Pattern in Fibroblasts from Arterial Tortuosity Syndrome Patients.

Ascorbate requiring Fe2+/2-oxoglutarate-dependent dioxygenases located in the nucleoplasm have been shown to participate in epigenetic regulation of gene expression via histone and DNA demethylation. Transport of dehydroascorbic acid is impaired in the endomembranes of fibroblasts from arterial tortuosity syndrome (ATS) patients, due to the mutation in the gene coding for glucose transporter GLUT10. We hypothesized that altered nuclear ascorbate concentration might be present in ATS fibroblasts, affecting dioxygenase activity and DNA demethylation. Therefore, our aim was to characterize the subcellular distribution of vitamin C, the global and site-specific changes in 5-methylcytosine and 5-hydroxymethylcytosine levels, and the effect of ascorbate supplementation in control and ATS fibroblast cultures. Diminished nuclear accumulation of ascorbate was found in ATS fibroblasts upon ascorbate or dehydroascorbic acid addition. Analyzing DNA samples of cultured fibroblasts from controls and ATS patients, a lower global 5-hydroxymethylcytosine level was found in ATS fibroblasts, which could not be significantly modified by ascorbate addition. Investigation of the (hydroxy)methylation status of specific regions in six candidate genes related to ascorbate metabolism and function showed that ascorbate addition could stimulate hydroxymethylation and active DNA demethylation at the PPAR-γ gene region in control fibroblasts only. The altered DNA hydroxymethylation patterns in patient cells both at the global level and at specific gene regions accompanied with decreased nuclear accumulation of ascorbate suggests the epigenetic role of vitamin C in the pathomechanism of ATS. The present findings represent the first example for the role of vitamin C transport in epigenetic regulation suggesting that ATS is a compartmentalization disease.

Cellular toxicity of dietary trans fatty acids and its correlation with ceramide and diglyceride accumulation.

High fatty acid (FA) levels are deleterious to pancreatic ß-cells, largely due to the accumulation of biosynthetic lipid intermediates, such as ceramides and diglycerides, which induce ER stress and apoptosis. Toxicity of palmitate (16:0) and oleate (18:1 cis-Δ9) has been widely investigated, while very little data is available on the cell damages caused by elaidate (18:1 trans-Δ9) and vaccenate (18:1 trans-Δ11), although the potential health effects of these dietary trans fatty acids (TFAs) received great publicity. We compared the effects of these four FAs on cell viability, apoptosis, ER stress, JNK phosphorylation and autophagy as well as on ceramide and diglyceride contents in RINm5F insulinoma cells. Similarly to oleate and unlike palmitate, TFAs reduced cell viability only at higher concentration, and they had mild effects on ER stress, apoptosis and autophagy. Palmitate increased ceramide and diglyceride levels far more than any of the unsaturated fatty acids; however, incorporation of TFAs in ceramides and diglycerides was strikingly more pronounced than that of oleate. This indicates a correlation between the accumulation of lipid intermediates and the severity of cell damage. Our findings reveal important metabolic characteristics of TFAs that might underlie a long term toxicity and hence deserve further investigation.

Cerebrospinal Fluid-Contacting Neurons Sense pH Changes and Motion in the Hypothalamus.

CSF-contacting (CSF-c) cells are present in the walls of the brain ventricles and the central canal of the spinal cord and found throughout the vertebrate phylum. We recently identified ciliated somatostatin-/GABA-expressing CSF-c neurons in the lamprey spinal cord that act as pH sensors as well as mechanoreceptors. In the same neuron, acidic and alkaline responses are mediated through ASIC3-like and PKD2L1 channels, respectively. Here, we investigate the functional properties of the ciliated somatostatin-/GABA-positive CSF-c neurons in the hypothalamus by performing whole-cell recordings in hypothalamic slices. Depolarizing current pulses readily evoked action potentials, but hypothalamic CSF-c neurons had no or a very low level of spontaneous activity at pH 7.4. They responded, however, with membrane potential depolarization and trains of action potentials to small deviations in pH in both the acidic and alkaline direction. Like in spinal CSF-c neurons, the acidic response in hypothalamic cells is mediated via ASIC3-like channels. In contrast, the alkaline response appears to depend on connexin hemichannels, not on PKD2L1 channels. We also show that hypothalamic CSF-c neurons respond to mechanical stimulation induced by fluid movements along the wall of the third ventricle, a response mediated via ASIC3-like channels. The hypothalamic CSF-c neurons extend their processes dorsally, ventrally, and laterally, but as yet, the effects exerted on hypothalamic circuits are unknown. With similar neurons being present in rodents, the pH- and mechanosensing ability of hypothalamic CSF-c neurons is most likely conserved throughout vertebrate phylogeny.SIGNIFICANCE STATEMENT CSF-contacting neurons are present in all vertebrates and are located mainly in the hypothalamic area and the spinal cord. Here, we report that the somatostatin-/GABA-expressing CSF-c neurons in the lamprey hypothalamus sense bidirectional deviations in the extracellular pH and do so via different molecular mechanisms. They also serve as mechanoreceptors. The hypothalamic CSF-c neurons have extensive axonal ramifications and may decrease the level of motor activity via release of somatostatin. In conclusion, hypothalamic somatostatin-/GABA-expressing CSF-c neurons, as well as their spinal counterpart, represent a novel homeostatic mechanism designed to sense any deviation from physiological pH and thus constitute a feedback regulatory system intrinsic to the CNS, possibly serving a protective role from damage caused by changes in pH.

Ca2+-binding protein NECAB2 facilitates inflammatory pain hypersensitivity.

Pain signals are transmitted by multisynaptic glutamatergic pathways. Their first synapse between primary nociceptors and excitatory spinal interneurons gates the sensory load. In this pathway, glutamate release is orchestrated by Ca2+-sensor proteins, with N-terminal EF-hand Ca2+-binding protein 2 (NECAB2) being particular abundant. However, neither the importance of NECAB2+ neuronal contingents in dorsal root ganglia (DRGs) and spinal cord nor the function determination by NECAB2 has been defined. A combination of histochemical analyses and single-cell RNA-sequencing showed NECAB2 in small- and medium-sized C- and Aδ D-hair low-threshold mechanoreceptors in DRGs, as well as in protein kinase C γ excitatory spinal interneurons. NECAB2 was downregulated by peripheral nerve injury, leading to the hypothesis that NECAB2 loss of function could limit pain sensation. Indeed, Necab2-/- mice reached a pain-free state significantly faster after peripheral inflammation than did WT littermates. Genetic access to transiently activated neurons revealed that a mediodorsal cohort of NECAB2+ neurons mediates inflammatory pain in the mouse spinal dorsal horn. Here, besides dampening excitatory transmission in spinal interneurons, NECAB2 limited pronociceptive brain-derived neurotrophic factor (BDNF) release from sensory afferents. Hoxb8-dependent reinstatement of NECAB2 expression in Necab2-/- mice then demonstrated that spinal and DRG NECAB2 alone could control inflammation-induced sensory hypersensitivity. Overall, we identify NECAB2 as a critical component of pronociceptive pain signaling, whose inactivation offers substantial pain relief.