Tumor and Microenvironment Evolution during Immunotherapy with Nivolumab.

The mechanisms by which immune checkpoint blockade modulates tumor evolution during therapy are unclear. We assessed genomic changes in tumors from 68 patients with advanced melanoma, who progressed on ipilimumab or were ipilimumab-naive, before and after nivolumab initiation (CA209-038 study). Tumors were analyzed by whole-exome, transcriptome, and/or T cell receptor (TCR) sequencing. In responding patients, mutation and neoantigen load were reduced from baseline, and analysis of intratumoral heterogeneity during therapy demonstrated differential clonal evolution within tumors and putative selection against neoantigenic mutations on-therapy. Transcriptome analyses before and during nivolumab therapy revealed increases in distinct immune cell subsets, activation of specific transcriptional networks, and upregulation of immune checkpoint genes that were more pronounced in patients with response. Temporal changes in intratumoral TCR repertoire revealed expansion of T cell clones in the setting of neoantigen loss. Comprehensive genomic profiling data in this study provide insight into nivolumab's mechanism of action.

Inhibiting DNA Methylation Causes an Interferon Response in Cancer via dsRNA Including Endogenous Retroviruses.

We show that DNA methyltransferase inhibitors (DNMTis) upregulate immune signaling in cancer through the viral defense pathway. In ovarian cancer (OC), DNMTis trigger cytosolic sensing of double-stranded RNA (dsRNA) causing a type I interferon response and apoptosis. Knocking down dsRNA sensors TLR3 and MAVS reduces this response 2-fold and blocking interferon beta or its receptor abrogates it. Upregulation of hypermethylated endogenous retrovirus (ERV) genes accompanies the response and ERV overexpression activates the response. Basal levels of ERV and viral defense gene expression significantly correlate in primary OC and the latter signature separates primary samples for multiple tumor types from The Cancer Genome Atlas into low versus high expression groups. In melanoma patients treated with an immune checkpoint therapy, high viral defense signature expression in tumors significantly associates with durable clinical response and DNMTi treatment sensitizes to anti-CTLA4 therapy in a pre-clinical melanoma model.

H3K9 methylation drives resistance to androgen receptor-antagonist therapy in prostate cancer.

Antiandrogen strategies remain the prostate cancer treatment backbone, but drug resistance develops. We show that androgen blockade in prostate cancer leads to derepression of retroelements (REs) followed by a double-stranded RNA (dsRNA)-stimulated interferon response that blocks tumor growth. A forward genetic approach identified H3K9 trimethylation (H3K9me3) as an essential epigenetic adaptation to antiandrogens, which enabled transcriptional silencing of REs that otherwise stimulate interferon signaling and glucocorticoid receptor expression. Elevated expression of terminal H3K9me3 writers was associated with poor patient hormonal therapy outcomes. Forced expression of H3K9me3 writers conferred resistance, whereas inhibiting H3K9-trimethylation writers and readers restored RE expression, blocking antiandrogen resistance. Our work reveals a drug resistance axis that integrates multiple cellular signaling elements and identifies potential pharmacologic vulnerabilities.

A neoantigen fitness model predicts tumour response to checkpoint blockade immunotherapy.

Checkpoint blockade immunotherapies enable the host immune system to recognize and destroy tumour cells. Their clinical activity has been correlated with activated T-cell recognition of neoantigens, which are tumour-specific, mutated peptides presented on the surface of cancer cells. Here we present a fitness model for tumours based on immune interactions of neoantigens that predicts response to immunotherapy. Two main factors determine neoantigen fitness: the likelihood of neoantigen presentation by the major histocompatibility complex (MHC) and subsequent recognition by T cells. We estimate these components using the relative MHC binding affinity of each neoantigen to its wild type and a nonlinear dependence on sequence similarity of neoantigens to known antigens. To describe the evolution of a heterogeneous tumour, we evaluate its fitness as a weighted effect of dominant neoantigens in the subclones of the tumour. Our model predicts survival in anti-CTLA-4-treated patients with melanoma and anti-PD-1-treated patients with lung cancer. Importantly, low-fitness neoantigens identified by our method may be leveraged for developing novel immunotherapies. By using an immune fitness model to study immunotherapy, we reveal broad similarities between the evolution of tumours and rapidly evolving pathogens.

Identification of unique neoantigen qualities in long-term survivors of pancreatic cancer.

Pancreatic ductal adenocarcinoma is a lethal cancer with fewer than 7% of patients surviving past 5 years. T-cell immunity has been linked to the exceptional outcome of the few long-term survivors, yet the relevant antigens remain unknown. Here we use genetic, immunohistochemical and transcriptional immunoprofiling, computational biophysics, and functional assays to identify T-cell antigens in long-term survivors of pancreatic cancer. Using whole-exome sequencing and in silico neoantigen prediction, we found that tumours with both the highest neoantigen number and the most abundant CD8+ T-cell infiltrates, but neither alone, stratified patients with the longest survival. Investigating the specific neoantigen qualities promoting T-cell activation in long-term survivors, we discovered that these individuals were enriched in neoantigen qualities defined by a fitness model, and neoantigens in the tumour antigen MUC16 (also known as CA125). A neoantigen quality fitness model conferring greater immunogenicity to neoantigens with differential presentation and homology to infectious disease-derived peptides identified long-term survivors in two independent datasets, whereas a neoantigen quantity model ascribing greater immunogenicity to increasing neoantigen number alone did not. We detected intratumoural and lasting circulating T-cell reactivity to both high-quality and MUC16 neoantigens in long-term survivors of pancreatic cancer, including clones with specificity to both high-quality neoantigens and predicted cross-reactive microbial epitopes, consistent with neoantigen molecular mimicry. Notably, we observed selective loss of high-quality and MUC16 neoantigenic clones on metastatic progression, suggesting neoantigen immunoediting. Our results identify neoantigens with unique qualities as T-cell targets in pancreatic ductal adenocarcinoma. More broadly, we identify neoantigen quality as a biomarker for immunogenic tumours that may guide the application of immunotherapies.

Effects of pulsed electric fields on exciton propagation efficiency along Müller cell intermediate filaments. Possible separation mechanism of high- and low-contrast images by the eye-brain system.

In the current study, we tested a possible mechanism of low- and high-contrast image component discrimination by the vertebrate eye-brain system. Apparently the eye-brain system has to discriminate between the low-contrast image component formed by light scattered within the retina, due to interaction of photons with cells and their parts, and the high-contrast image component transmitted by excitons via the quantum mechanism. Presently, effects of pulsed electric fields applied to Müller cell (MC) intermediate filaments (IFs) on the efficiency of exciton propagation were explored. The effects of both pulse duration and amplitude were recorded. These experimental results show that the eye-brain system may be using signal modulation to discriminate between high- and low-contrast image components, improving our understanding of high-contrast vision in vertebrates.

Comparison of EMT-Related and Multi-Drug Resistant Gene Expression, Extracellular Matrix Production, and Drug Sensitivity in NSCLC Spheroids Generated by Scaffold-Free and Scaffold-Based Methods.

Multicellular 3D tumor models are becoming a powerful tool for testing of novel drug products and personalized anticancer therapy. Tumor spheroids, a commonly used 3D multicellular tumor model, more closely reproduce the tumor microenvironment than conventional 2D cell cultures. It should be noted that spheroids can be produced using different techniques, which can be subdivided into scaffold-free (SF) and scaffold-based (SB) methods. However, it remains unclear, to what extent spheroid properties depend on the method of their generation. In this study, we aimed to carry out a head-to-head comparison of drug sensitivity and molecular expression profile in SF and SB spheroids along with a monolayer (2D) cell culture. Here, we produced non-small cell lung cancer (NSCLC) spheroids based on human lung adenocarcinoma cell line A549. Drug sensitivity analysis of the tested cell cultures to five different chemotherapeutics resulted in IC50 (A549-SB) > IC50 (A549-SF) > IC50 (A549-2D) trend. It was found that SF and SB A549 spheroids displayed elevated expression levels of epithelial-to-mesenchymal transition (EMT) markers and proteins associated with drug resistance compared with the monolayer A549 cell culture. Enhanced drug resistance of A549-SB spheroids can be a result of larger diameters and elevated deposition of extracellular matrix (ECM) that impairs drug penetration into spheroids. Thus, the choice of the spheroid production method can influence the properties of the generated 3D cell culture and their drug resistance. This fact should be considered for correct interpretation of drug testing results.

New unique optical and electric properties of intermediate filaments in Müller cells.

Presently we report new unique optical and electric properties of Müller cell (MC) intermediate filaments (IFs). We inform that these IFs extracted from porcine retina are excellent conductors of light and electric current. Such IF properties may endow vertebrate eyes with high-contrast vision. The properties of the IFs allow a simple quantum-mechanical description that justifies the quantum mechanism (QM) for the light energy transfer between the inner and the outer limiting membranes. These properties also provide direct and unequivocal proof that QM works even in isolated IFs, while the classic theory admits no capacity for light transmission by objects that are so thin. Note that the length and diameter of the IFs were 117 ±â€¯1.3 µm and 10.1 ±â€¯0.07 nm, respectively. The QM avoids the light scattering effects, which could significantly reduce the visual contrast, by conducting light energy in the form of excitons (excited states). This scientific breakthrough may provide new insights for medical ophthalmology.

Simple kinetic model of mitochondrial swelling in cardiac cells.

Mitochondria play an important role in both cell survival and cell death. In response to oxidative stress, they undergo opening of non-selective permeability transition pores (PTP) in the inner mitochondrial membrane. Sustained PTP opening triggers mitochondrial swelling due to increased colloidal osmotic pressure in the matrix accompanied by mitochondrial membrane depolarization and ATP hydrolysis. Mitochondrial swelling is the major factor leading to mitochondria-mediated cell death through both apoptosis and necrosis. Hence, precise estimation of the threshold parameters of the transition of reversible swelling to irreversible swelling is important for understanding the mechanisms of PTP-mediated cell death as well as for the development of new therapeutic approaches targeting the mitochondria under pathological conditions. In this study, we designed a simple kinetic model of the Ca2+ -induced mitochondrial swelling that describes the mechanisms of transition from reversible to irreversible swelling in cardiac mitochondria. Values of kinetic parameters calculated using parameter estimation techniques that fit experimental data of mitochondrial swelling with minimum average differences between the experimental data and model parameters. Overall, this study provides a kinetic model verified by data simulation and model fitting that adequately describes the dynamics of mitochondrial swelling.

"Terhune-like" transformation of the terahertz polarization ellipse "mutually induced" by three-wave joint propagation in liquid.

In this Letter, we show experimentally for the first time, to the best of our knowledge, the possibility to observe the effect of polarization mutual action of three elliptically polarized waves, with one of them at terahertz frequency, when they propagate in the isotropic nonlinear medium. When three light pulses are propagated at frequencies ω, 2ω, and ωTHz through liquid nitrogen, we observed the rotation of the ellipse main axis and the ellipticity change. We have shown that this effect is very well described theoretically in the framework of a physical approach analogous to the self-rotation of the polarization ellipse first described in 1964 by Maker et al., but expanded for the case of multi-frequency interaction.

Müller glial cells contribute to dim light vision in the spectacled caiman (Caiman crocodilus fuscus): Analysis of retinal light transmission.

In this study, we show the capability of Müller glial cells to transport light through the inverted retina of reptiles, specifically the retina of the spectacled caimans. Thus, confirming that Müller cells of lower vertebrates also improve retinal light transmission. Confocal imaging of freshly isolated retinal wholemounts, that preserved the refractive index landscape of the tissue, indicated that the retina of the spectacled caiman is adapted for vision under dim light conditions. For light transmission experiments, we used a setup with two axially aligned objectives imaging the retina from both sides to project the light onto the inner (vitreal) surface and to detect the transmitted light behind the retina at the receptor layer. Simultaneously, a confocal microscope obtained images of the Müller cells embedded within the vital tissue. Projections of light onto several representative Müller cell trunks within the inner plexiform layer, i.e. (i) trunks with a straight orientation, (ii) trunks which are formed by the inner processes and (iii) trunks which get split into inner processes, were associated with increases in the intensity of the transmitted light. Projections of light onto the periphery of the Müller cell endfeet resulted in a lower intensity of transmitted light. In this way, retinal glial (Müller) cells support dim light vision by improving the signal-to-noise ratio which increases the sensitivity to light. The field of illuminated photoreceptors mainly include rods reflecting the rod dominance of the of tissue. A subpopulation of Müller cells with downstreaming cone cells led to a high-intensity illumination of the cones, while the surrounding rods were illuminated by light of lower intensity. Therefore, Müller cells that lie in front of cones may adapt the intensity of the transmitted light to the different sensitivities of cones and rods, presumably allowing a simultaneous vision with both receptor types under dim light conditions.

Patterns and rates of exonic de novo mutations in autism spectrum disorders.

Autism spectrum disorders (ASD) are believed to have genetic and environmental origins, yet in only a modest fraction of individuals can specific causes be identified. To identify further genetic risk factors, here we assess the role of de novo mutations in ASD by sequencing the exomes of ASD cases and their parents (n = 175 trios). Fewer than half of the cases (46.3%) carry a missense or nonsense de novo variant, and the overall rate of mutation is only modestly higher than the expected rate. In contrast, the proteins encoded by genes that harboured de novo missense or nonsense mutations showed a higher degree of connectivity among themselves and to previous ASD genes as indexed by protein-protein interaction screens. The small increase in the rate of de novo events, when taken together with the protein interaction results, are consistent with an important but limited role for de novo point mutations in ASD, similar to that documented for de novo copy number variants. Genetic models incorporating these data indicate that most of the observed de novo events are unconnected to ASD; those that do confer risk are distributed across many genes and are incompletely penetrant (that is, not necessarily sufficient for disease). Our results support polygenic models in which spontaneous coding mutations in any of a large number of genes increases risk by 5- to 20-fold. Despite the challenge posed by such models, results from de novo events and a large parallel case-control study provide strong evidence in favour of CHD8 and KATNAL2 as genuine autism risk factors.

Artificial neural network detects human uncertainty.

Artificial neural networks (ANNs) are known to be a powerful tool for data analysis. They are used in social science, robotics, and neurophysiology for solving tasks of classification, forecasting, pattern recognition, etc. In neuroscience, ANNs allow the recognition of specific forms of brain activity from multichannel EEG or MEG data. This makes the ANN an efficient computational core for brain-machine systems. However, despite significant achievements of artificial intelligence in recognition and classification of well-reproducible patterns of neural activity, the use of ANNs for recognition and classification of patterns in neural networks still requires additional attention, especially in ambiguous situations. According to this, in this research, we demonstrate the efficiency of application of the ANN for classification of human MEG trials corresponding to the perception of bistable visual stimuli with different degrees of ambiguity. We show that along with classification of brain states associated with multistable image interpretations, in the case of significant ambiguity, the ANN can detect an uncertain state when the observer doubts about the image interpretation. With the obtained results, we describe the possible application of ANNs for detection of bistable brain activity associated with difficulties in the decision-making process.

Computational Modeling of In Vitro Swelling of Mitochondria: A Biophysical Approach.

Swelling of mitochondria plays an important role in the pathogenesis of human diseases by stimulating mitochondria-mediated cell death through apoptosis, necrosis, and autophagy. Changes in the permeability of the inner mitochondrial membrane (IMM) of ions and other substances induce an increase in the colloid osmotic pressure, leading to matrix swelling. Modeling of mitochondrial swelling is important for simulation and prediction of in vivo events in the cell during oxidative and energy stress. In the present study, we developed a computational model that describes the mechanism of mitochondrial swelling based on osmosis, the rigidity of the IMM, and dynamics of ionic/neutral species. The model describes a new biophysical approach to swelling dynamics, where osmotic pressure created in the matrix is compensated for by the rigidity of the IMM, i.e., osmotic pressure induces membrane deformation, which compensates for the osmotic pressure effect. Thus, the effect is linear and reversible at small membrane deformations, allowing the membrane to restore its normal form. On the other hand, the membrane rigidity drops to zero at large deformations, and the swelling becomes irreversible. As a result, an increased number of dysfunctional mitochondria can activate mitophagy and initiate cell death. Numerical modeling analysis produced results that reasonably describe the experimental data reported earlier.

SCN4A pore mutation pathogenetically contributes to autosomal dominant essential tremor and may increase susceptibility to epilepsy.

Essential tremor (ET) is the most prevalent movement disorder, affecting millions of people in the USA. Although a positive family history is one of the most important risk factors for ET, the genetic causes of ET remain unknown. In an attempt to identify genetic causes for ET, we performed whole-exome sequencing analyses in a large Spanish family with ET, in which two patients also developed epilepsy. To further assess pathogenicity, site-directed mutagenesis, mouse and human brain expression analyses, and patch clamp techniques were performed. A disease-segregating mutation (p.Gly1537Ser) in the SCN4A gene was identified. Posterior functional analyses demonstrated that more rapid kinetics at near-threshold potentials altered ion selectivity and facilitated the conductance of both potassium and ammonium ions, which could contribute to tremor and increase susceptibility to epilepsy, respectively. In this report, for the first time, we associated the genetic variability of SCN4A with the development of essential tremor, which adds ET to the growing list of neurological channelopathies.

Genetic basis for clinical response to CTLA-4 blockade in melanoma.

BACKGROUND: Immune checkpoint inhibitors are effective cancer treatments, but molecular determinants of clinical benefit are unknown. Ipilimumab and tremelimumab are antibodies against cytotoxic T-lymphocyte antigen 4 (CTLA-4). Anti-CTLA-4 treatment prolongs overall survival in patients with melanoma. CTLA-4 blockade activates T cells and enables them to destroy tumor cells. METHODS: We obtained tumor tissue from patients with melanoma who were treated with ipilimumab or tremelimumab. Whole-exome sequencing was performed on tumors and matched blood samples. Somatic mutations and candidate neoantigens generated from these mutations were characterized. Neoantigen peptides were tested for the ability to activate lymphocytes from ipilimumab-treated patients. RESULTS: Malignant melanoma exomes from 64 patients treated with CTLA-4 blockade were characterized with the use of massively parallel sequencing. A discovery set consisted of 11 patients who derived a long-term clinical benefit and 14 patients who derived a minimal benefit or no benefit. Mutational load was associated with the degree of clinical benefit (P=0.01) but alone was not sufficient to predict benefit. Using genomewide somatic neoepitope analysis and patient-specific HLA typing, we identified candidate tumor neoantigens for each patient. We elucidated a neoantigen landscape that is specifically present in tumors with a strong response to CTLA-4 blockade. We validated this signature in a second set of 39 patients with melanoma who were treated with anti-CTLA-4 antibodies. Predicted neoantigens activated T cells from the patients treated with ipilimumab. CONCLUSIONS: These findings define a genetic basis for benefit from CTLA-4 blockade in melanoma and provide a rationale for examining exomes of patients for whom anti-CTLA-4 agents are being considered. (Funded by the Frederick Adler Fund and others.).

The role of neoantigens in response to immune checkpoint blockade.

Immune checkpoint blockade has demonstrated substantial promise for the treatment of several advanced malignancies. These agents activate the immune system to attack tumor cells. For example, agents targeting CTLA4 and programmed cell death 1 (PD-1) have resulted in impressive response rates and, in some cases, durable remissions. Neoantigens are mutations that encode immunologically active proteins that can cause the immune system to recognize the affected cell as foreign. Recent data have made it clear that these mutations are, in large part, the functional targets of immune checkpoint blockade. This review summarizes the key discoveries leading up to this important conclusion and discusses possible applications of neoantigens in cancer therapy.

Duplications of the neuropeptide receptor gene VIPR2 confer significant risk for schizophrenia.

Rare copy number variants (CNVs) have a prominent role in the aetiology of schizophrenia and other neuropsychiatric disorders. Substantial risk for schizophrenia is conferred by large (>500-kilobase) CNVs at several loci, including microdeletions at 1q21.1 (ref. 2), 3q29 (ref. 3), 15q13.3 (ref. 2) and 22q11.2 (ref. 4) and microduplication at 16p11.2 (ref. 5). However, these CNVs collectively account for a small fraction (2-4%) of cases, and the relevant genes and neurobiological mechanisms are not well understood. Here we performed a large two-stage genome-wide scan of rare CNVs and report the significant association of copy number gains at chromosome 7q36.3 with schizophrenia. Microduplications with variable breakpoints occurred within a 362-kilobase region and were detected in 29 of 8,290 (0.35%) patients versus 2 of 7,431 (0.03%) controls in the combined sample. All duplications overlapped or were located within 89 kilobases upstream of the vasoactive intestinal peptide receptor gene VIPR2. VIPR2 transcription and cyclic-AMP signalling were significantly increased in cultured lymphocytes from patients with microduplications of 7q36.3. These findings implicate altered vasoactive intestinal peptide signalling in the pathogenesis of schizophrenia and indicate the VPAC2 receptor as a potential target for the development of new antipsychotic drugs.