Clonal KEAP1 mutations with loss of heterozygosity share reduced immunotherapy efficacy and low immune cell infiltration in lung adenocarcinoma.

BACKGROUND: KEAP1 mutations have been associated with reduced survival in lung adenocarcinoma (LUAD) patients treated with immune checkpoint inhibitors (ICIs), particularly in the presence of STK11/KRAS alterations. We hypothesized that, beyond co-occurring genomic events, clonality prediction may help identify deleterious KEAP1 mutations and their counterparts with retained sensitivity to ICIs. PATIENTS AND METHODS: Beta-binomial modelling of sequencing read counts was used to infer KEAP1 clonal inactivation by combined somatic mutation and loss of heterozygosity (KEAP1 C-LOH) versus partial inactivation [KEAP1 clonal diploid-subclonal (KEAP1 CD-SC)] in the Memorial Sloan Kettering Cancer Center (MSK) MetTropism cohort (N = 2550). Clonality/LOH prediction was compared to a streamlined clinical classifier that relies on variant allele frequencies (VAFs) and tumor purity (TP) (VAF/TP ratio). The impact of this classification on survival outcomes was tested in two independent cohorts of LUAD patients treated with immunotherapy (MSK/Rome N = 237; DFCI N = 461). Immune-related features were studied by exploiting RNA-sequencing data (TCGA) and multiplexed immunofluorescence (DFCI mIF cohort). RESULTS: Clonality/LOH inference in the MSK MetTropism cohort overlapped with a clinical classification model defined by the VAF/TP ratio. In the ICI-treated MSK/Rome discovery cohort, predicted KEAP1 C-LOH mutations were associated with shorter progression-free survival (PFS) and overall survival (OS) compared to KEAP1 wild-type cases (PFS log-rank P = 0.001; OS log-rank P < 0.001). Similar results were obtained in the DFCI validation cohort (PFS log-rank P = 0.006; OS log-rank P = 0.014). In both cohorts, we did not observe any significant difference in survival outcomes when comparing KEAP1 CD-SC and wild-type tumors. Immune deconvolution and multiplexed immunofluorescence revealed that KEAP1 C-LOH and KEAP1 CD-SC differed for immune-related features. CONCLUSIONS: KEAP1 C-LOH mutations are associated with an immune-excluded phenotype and worse clinical outcomes among advanced LUAD patients treated with ICIs. By contrast, survival outcomes of patients whose tumors harbored KEAP1 CD-SC mutations were similar to those with KEAP1 wild-type LUADs.

KEAP1-driven co-mutations in lung adenocarcinoma unresponsive to immunotherapy despite high tumor mutational burden.

BACKGROUND: Immune checkpoint inhibitors (ICIs) have demonstrated significant overall survival (OS) benefit in lung adenocarcinoma (LUAD). Nevertheless, a remarkable interpatient heterogeneity characterizes immunotherapy efficacy, regardless of programmed death-ligand 1 (PD-L1) expression and tumor mutational burden (TMB). KEAP1 mutations are associated with shorter survival in LUAD patients receiving chemotherapy. We hypothesized that the pattern of KEAP1 co-mutations and mutual exclusivity may identify LUAD patients unresponsive to immunotherapy. PATIENTS AND METHODS: KEAP1 mutational co-occurrences and somatic interactions were studied in the whole MSKCC LUAD dataset. The impact of coexisting alterations on survival outcomes in ICI-treated LUAD patients was verified in the randomized phase II/III POPLAR/OAK trials (blood-based sequencing, bNGS cohort, N = 253). Three tissue-based sequencing studies (Rome, MSKCC and DFCI) were used for independent validation (tNGS cohort, N = 289). Immunogenomic features were analyzed using The Cancer Genome Atlas (TCGA) LUAD study. RESULTS: On the basis of KEAP1 mutational co-occurrences, we identified four genes potentially associated with reduced efficacy of immunotherapy (KEAP1, PBRM1, SMARCA4 and STK11). Independent of the nature of co-occurring alterations, tumors with coexisting mutations (CoMut) had inferior survival as compared with single-mutant (SM) and wild-type (WT) tumors (bNGS cohort: CoMut versus SM log-rank P = 0.048, CoMut versus WT log-rank P < 0.001; tNGS cohort: CoMut versus SM log-rank P = 0.037, CoMut versus WT log-rank P = 0.006). The CoMut subset harbored higher TMB than the WT disease and the adverse significance of coexisting alterations was maintained in LUAD with high TMB. Significant immunogenomic differences were observed between the CoMut and WT groups in terms of core immune signatures, T-cell receptor repertoire, T helper cell signatures and immunomodulatory genes. CONCLUSIONS: This study indicates that coexisting alterations in a limited set of genes characterize a subset of LUAD unresponsive to immunotherapy and with high TMB. An immune-cold microenvironment may account for the clinical course of the disease.

Radial Spin Texture of the Weyl Fermions in Chiral Tellurium.

Trigonal tellurium, a small-gap semiconductor with pronounced magneto-electric and magneto-optical responses, is among the simplest realizations of a chiral crystal. We have studied by spin- and angle-resolved photoelectron spectroscopy its unconventional electronic structure and unique spin texture. We identify Kramers-Weyl, composite, and accordionlike Weyl fermions, so far only predicted by theory, and show that the spin polarization is parallel to the wave vector along the lines in k space connecting high-symmetry points. Our results clarify the symmetries that enforce such spin texture in a chiral crystal, thus bringing new insight in the formation of a spin vectorial field more complex than the previously proposed hedgehog configuration. Our findings thus pave the way to a classification scheme for these exotic spin textures and their search in chiral crystals.

Selective Probing of Hidden Spin-Polarized States in Inversion-Symmetric Bulk MoS_{2}.

Spin- and angle-resolved photoemission spectroscopy is used to reveal that a large spin polarization is observable in the bulk centrosymmetric transition metal dichalcogenide MoS_{2}. It is found that the measured spin polarization can be reversed by changing the handedness of incident circularly polarized light. Calculations based on a three-step model of photoemission show that the valley and layer-locked spin-polarized electronic states can be selectively addressed by circularly polarized light, therefore providing a novel route to probe these hidden spin-polarized states in inversion-symmetric systems as predicted by Zhang et al. [Nat. Phys. 10, 387 (2014).NPAHAX1745-247310.1038/nphys2933].

Evidence of trigonal dangling bonds at the Ge(111)/oxide interface by electrically detected magnetic resonance.

Despite a renewed interest in Ge as a competitor with Si for a broad range of electronic applications, the microstructure and the electronic properties of the dangling bonds that, in analogy with Si, are expected at the Ge/oxide interface have escaped a firm spectroscopy observation and characterization. Clear evidence based on contactless electrically detected magnetic resonance spectroscopy of a dangling bond at the Ge(111)/GeO(2) interface is reported in this Letter. This result supports the similarity between dangling bonds at the Si(111)/oxide and Ge(111)/oxide interfaces, both showing C(3v) trigonal point symmetry with the main axis oriented along the ⟨111⟩ direction. In contrast, at the Ge(001)/oxide interface the absence of the trigonal center in favor of a lower symmetry dangling bond marks the difference with the Si(001)/oxide interface, where both centers are present and the one having higher point symmetry prevails. This fact is rationalized in terms of suboxide interface rearrangement and oxide viscoelasticity, which promote the generation of the nonaxial centers at distorted dimers. The unambiguous identification of the centers at the Ge/oxide interfaces yields a deeper insight into the physical properties of the suboxide interface structure and offers a valid indicator for the evaluation of different surface capping and passivation techniques, with the potential to boost the Ge-related technology.

AATF/Che-1 RNA polymerase II binding protein overexpression reduces the anti-tumor NK-cell cytotoxicity through activating receptors modulation.

Introduction: AATF/Che-1 over-expression in different tumors is well known and its effect on tumorigenicity is mainly due to its central role demonstrated in the oncogenic pathways of solid tumors, where it controls proliferation and viability. The effect exerted by tumors overexpressing Che-1 on the immune response has not yet been investigated. Methods: Starting from ChIP-sequencing data we confirmed Che-1 enrichment on Nectin-1 promoter. Several co-cultures experiments between NK-cells and tumor cells transduced by lentiviral vectors carrying Che-1-interfering sequence, analyzed by flow-cytometry have allowed a detailed characterization of NK receptors and tumor ligands expression. Results: Here, we show that Che-1 is able to modulate the expression of Nectin-1 ligand at the transcriptional level, leading to the impairment of killing activity of NK-cells. Nectin-1 down-modulation induces a modification in NK-cell ligands expression able to interact with activating receptors and to stimulate NK-cell function. In addition, NK-cells from Che-1 transgenic mice, confirming a reduced expression of activating receptors, exhibit impaired activation and a preferential immature status. Discussion: The critical equilibrium between NK-cell ligand expression on tumor cells and the interaction with NK cell receptors is affected by Che-1 over-expression and partially restored by Che-1 interference. The evidence of a new role for Che-1 as regulator of anti-tumor immunity supports the necessity to develop approaches able to target this molecule which shows a dual tumorigenic function as cancer promoter and immune response modulator.

Significant enhancement of ferromagnetism above room temperature in epitaxial 2D van der Waals ferromagnet Fe5-δGeTe2/Bi2Te3 heterostructures.

Two-dimensional (2D) van der Waals (vdW) ferromagnetic metals FexGeTe2 with x = 3-5 have raised significant interest in the scientific community. Fe5GeTe2 shows prospects for spintronic applications since the Curie temperature Tc has been reported near or higher than 300 K. In the present work, epitaxial Fe5-δGeTe2 (FGT) heterostructures were grown by Molecular Beam Epitaxy (MBE) on insulating crystalline substrates. The FGT films were combined with Bi2Te3 topological insulator (TI) aiming to investigate the possible beneficial effect of the TI on the magnetic properties of FGT. FGT/Bi2Te3 films were compared to FGT capped only with AlOx to prevent oxidation. SQUID and MOKE measurements revealed that the growth of Bi2Te3 TI on FGT films significantly enhances the saturation magnetization of FGT as well as the Tc well above room temperature (RT) reaching record values of 570 K. First-principles calculations predict a shift of the Fermi level and an associated enhancement of the majority spin (primarily) as well as the total density of states at the Fermi level suggesting that effective doping of FGT from Bi2Te3 could explain the enhancement of ferromagnetism in FGT. It is also predicted that strain induced stabilization of a high magnetic moment phase in FGT/Bi2Te3 could be an alternative explanation of magnetization and Tc enhancement. Ferromagnetic resonance measurements evidence an enhanced broadening in the FGT/Bi2Te3 heterostructure when compared to FGT. We obtain a large spin mixing conductance of g↑↓eff = 4.4 × 1020 m-2, which demonstrates the great potential of FGT/Bi2Te3 systems for spin-charge conversion applications at room temperature.

Electronic properties of pristine and Se doped [001] silicon nanowires: an ab initio study.

We report the results of first-principles density functional theory calculations of the properties of pristine and Se doped hydrogen passivated silicon nanowires oriented along the [001] direction. We show that the effect of confinement in the orthogonal directions is to increase the bandgap as the diameter of the nanowire is progressively reduced, as expected from the simple quantum confinement model. Moreover, we observe that the introduction of surface reconstruction before H passivation can lead to an indirect bandgap due to a shift of the valence band maximum away from the convergence point. We also study the formation energy and hyperfine parameters of Se located in different positions in a nanowire with diameter of 0.8 nm. Our results confirms a tendency to surface segregation for Se atoms, probably driven by deformations close to the nanowire surface. These deformations cause also a steep decrease in the hyperfine contact term of Se in surface positions compared to the situation when Se is placed near the axis of the nanowire. The latter values of the hyperfine contact term are instead much greater than the corresponding bulk values.

The fabrication of tunable nanoporous oxide surfaces by block copolymer lithography and atomic layer deposition.

Patterned nanoscale materials with controllable characteristic feature sizes and periodicity are of considerable interest in a wide range of fields, with various possible applications ranging from biomedical to nanoelectronic devices. Block-copolymer (BC)-based lithography is a powerful tool for the fabrication of uniform, densely spaced nanometer-scale features over large areas. Following this bottom-up approach, nanoporous polymeric films can be deposited on any type of substrate. The nanoporous periodic template can be transferred to the underlying substrate by dry anisotropic etching. Nevertheless the physical sizes of the polymeric mask represent an important limitation in the implementation of suitable lithographic protocols based on BC technology, since the diameter and the center-to-center distance of the pores cannot be varied independently in this class of materials. This problem could be overcome by combining block copolymer technology with atomic layer deposition (ALD): by means of BC-based lithography a nanoporous SiO2 template, with well-reproducible characteristic dimensions, can be fabricated and subsequently used as a backbone for the growth of perfectly conformal thin oxide films by ALD. In this work polystyrene-b-poly(methylmethacrylate) (PS-b-PMMA) BC and reactive ion etching are used to fabricate hexagonally packed 23 nm wide nanopores in a 50 nm thick SiO2 matrix. By ALD deposition of Al2O3 thin films onto the nanoporous SiO2 templates, nanostructured Al2O3 surfaces are obtained. By properly adjusting the thickness of the Al2O3 film the dimension of the pores in the oxide films is progressively reduced, with nanometer precision, from the original size down to complete filling of the pores, thus providing a simple and fast strategy for the fabrication of nanoporous Al2O3 surfaces with well-controllable feature size.

Gene copy number variation and common human disease.

Variation in gene copy number is increasingly recognized as a common, heritable source of inter-individual differences in genomic sequence. The role of copy number variation is well established in the pathogenesis of rare genomic disorders. More recently, germline and somatic copy number variation have been shown to be important pathogenic factors in a range of common diseases, including infectious, autoimmune and neuropsychiatric diseases and cancer. In this review, we describe the range of methods available for measuring copy number variants (CNVs) in individuals and populations, including the limitations of presently available assays, and highlight some key examples of common diseases in which CNVs have been shown clearly to have a pathogenic role. Although there has been major progress in this field in the last 5 years, understanding the full contribution of CNVs to the genetic basis of common diseases will require further studies, with more accurate CNV assays and larger cohorts than have presently been completed.

Si nanocrystal synthesis in HfO2/SiO/HfO2 multilayer structures.

The synthesis of two-dimensional arrays of Si nanocrystals in an HfO2 matrix has been achieved by deposition of HfO2/SiO/HfO2 multilayer structures followed by high temperature (1100 degrees C) thermal treatment in nitrogen atmosphere. Silicon out-diffusion from the SiO layer through the HfO2 films has been shown to be the limiting factor in the formation of the Si nanocrystals. Suitable strategies have been identified in order to overcome this limitation. Si nanocrystal formation has been achieved by properly adjusting the thickness of the SiO layer.

Che-1 activates XIAP expression in response to DNA damage.

X-linked inhibitor of apoptosis protein (XIAP) is a member of the inhibitor of apoptosis proteins family that selectively binds and inhibits caspase-3, -7 and -9. As such, XIAP is an extremely potent suppressor of apoptosis and an attractive target for cancer treatment. Che-1 is an antiapoptotic agent involved in the control of gene transcription and cell proliferation. Recently, we showed that the checkpoint kinases ATM/ATR and checkpoint kinase 2 physically and functionally interact with Che-1 and promote its phosphorylation and accumulation in response to DNA damage. These Che-1 modifications induce transcription of p53, and Che-1 depletion strongly sensitizes tumor cells to anticancer drugs. Here we show that Che-1 activates XIAP expression in response to DNA damage. This effect is mediated by Che-1 phosphorylation and requires NF-kappaB. Notably, we found that XIAP expression is necessary for antiapoptotic activity of Che-1 and that in vivo downregulation of Che-1 by small interference RNA strongly enhanced the cytotoxicity of anticancer drugs.

Copy number variation of Fc gamma receptor genes and disease predisposition.

Naturally occurring variation in gene copy number is increasingly recognized as a major source of inter-individual differences in phenotype and is an important susceptibility factor for genetically complex diseases. Several studies provide evidence of copy number variation at genes involved in inflammation and immunity highlighting their possible contribution to the inter-individual variation observed in immune responses. This review will explore copy number variation at the Fc gamma receptor cluster and its relevance in the pathogenesis of common human diseases.

Size dependence of the Mössbauer recoilless fraction in ß-Sn nanocrystals.

We study the size dependence of the Mössbauer recoilless fraction in ß-Sn nanocrystals by performing conversion electron Mössbauer spectroscopy at different temperatures. The Mössbauer recoilless fraction is intimately related to dynamical processes involving the nuclei during the γ-ray emission. The ß-Sn nanocrystals are embedded in a SiO(2) matrix, and they have a mean diameter ranging from 7 to 17 nm. A lowering of the recoilless fraction with decreasing cluster size is observed. The smallest nanocrystals reveal a 60% reduction of the recoilless fraction compared to the bulk value, while for the largest clusters we observe the same Mössbauer recoilless fraction as in the bulk. This suggests that the dynamical properties of the ß-Sn nanocrystals in SiO(2) approach those of the bulk for a critical mean diameter above 16 nm. The experimental results are compared with theoretical values obtained by a continuum model in which the relevant parameters are determined ab initio.

Atomic-scale study of the amorphous-to-crystalline phase transition mechanism in GeTe thin films.

The underlying mechanism driving the structural amorphous-to-crystalline transition in Group VI chalcogenides is still a matter of debate even in the simplest GeTe system. We exploit the extreme sensitivity of 57Fe emission Mössbauer spectroscopy, following dilute implantation of 57Mn (T½ = 1.5 min) at ISOLDE/CERN, to study the electronic charge distribution in the immediate vicinity of the 57Fe probe substituting Ge (FeGe), and to interrogate the local environment of FeGe over the amorphous-crystalline phase transition in GeTe thin films. Our results show that the local structure of as-sputtered amorphous GeTe is a combination of tetrahedral and defect-octahedral sites. The main effect of the crystallization is the conversion from tetrahedral to defect-free octahedral sites. We discover that only the tetrahedral fraction in amorphous GeTe participates to the change of the FeGe-Te chemical bonds, with a net electronic charge density transfer of ~ 1.6 e/a0 between FeGe and neighboring Te atoms. This charge transfer accounts for a lowering of the covalent character during crystallization. The results are corroborated by theoretical calculations within the framework of density functional theory. The observed atomic-scale chemical-structural changes are directly connected to the macroscopic phase transition and resistivity switch of GeTe thin films.

Diffusion reaction of oxygen in HfO2/SiO2/Si stacks.

We study the oxidation mechanism of silicon in the presence of a thin HfO2 layer. We performed a set of annealing in 18O2 atmosphere on HfO2/SiO2/Si stacks observing the 18O distribution in the SiO2 layer with time-of-flight secondary ion mass spectrometry (ToF-SIMS). The 18O distribution in HfO2/SiO2/Si stacks upon 18O2 annealing suggests that what is responsible for SiO2 growth is the molecular O2, whereas no contribution is found of the atomic oxygen to the oxidation. By studying the dependence of the oxidation velocity from oxygen partial pressure and annealing temperature, we demonstrate that the rate-determining step of the oxidation is the oxygen exchange at the HfO2/SiO2 interface. When moisture is chemisorbed in HfO2 films, the oxidation of the underlying silicon substrate becomes extremely fast and its kinetics can be described as a wet silicon oxidation process. The silicon oxidation during O2 annealing of the atomic layer deposited HfO2/Si is fast in its early stage due to chemisorbed moisture and becomes slow after the first 10 s.

Defective human retinoblastoma protein identified by lack of interaction with the E1A oncoprotein.

Inactivating mutations of the retinoblastoma susceptibility gene (Rb) are involved in the pathogenesis of hereditary and sporadic retinoblastoma. Alterations in the Rb gene have also been found in several other human tumors occurring with epidemiological incidence higher than that of retinoblastoma. Four human malignant glioma cell lines were examined for abnormalities in the retinoblastoma gene product (pRb), using a procedure based on the interaction of pRb with an in vitro-translated adenovirus E1A oncoprotein. In the CRS-A2 cell line, derived from a glioblastoma multiforme, pRb did not bind with the in vitro-translated E1A protein. Restriction analysis of the CRS-A2 Rb gene and Rb mRNA expression provided patterns that could not be distinguished from the other glioma cell lines. Further investigation revealed the presence of a truncated pRb in the CRS-A2 cell line, due to a nucleotide insertion in the coding sequence at position 2550. In addition, this truncated Rb protein was undetectable in phosphorylated form. The binding assay with the in vitro-translated E1A was also used to study other cell lines with known mutations in the Rb gene. This method, which evaluates the interaction between in vitro-translated E1A and the pRb, is proposed as a rapid screening for detecting functional alterations in the retinoblastoma protein.

Electrochemotherapy as First Line Cancer Treatment: Experiences from Veterinary Medicine in Developing Novel Protocols.

Tumor microenvironment is one of the major obstacles to the efficacy of chemotherapy in cancer patients. The abnormal blood flow within the tumor results in uneven drug distribution. Electrochemotherapy (ECT) is a tumor treatment that adopts the systemic or local delivery of anticancer drugs with the application of permeabilizing electric pulses having appropriate amplitude and waveforms. This allows the use of lipophobic drugs that frequently have a narrow therapeutic index maintaining at the same time a reduced patient morbidity and preserving appropriate anticancer efficacy. Its use in humans is addressed to the treatment of cutaneous neoplasms or the palliation of skin tumor metastases, and a standard operating procedure has been devised. On the other hand, in veterinary oncology this approach is gaining popularity, thus becoming a first line treatment for different cancer histotypes, in a variety of clinical conditions due to its high efficacy and low toxicity. This review summarizes the state of the art in veterinary oncology as a preclinical model and reports the new protocols in terms of drugs and therapy combination that have been developed.

Entanglement and manipulation of the magnetic and spin-orbit order in multiferroic Rashba semiconductors.

Entanglement of the spin-orbit and magnetic order in multiferroic materials bears a strong potential for engineering novel electronic and spintronic devices. Here, we explore the electron and spin structure of ferroelectric α-GeTe thin films doped with ferromagnetic Mn impurities to achieve its multiferroic functionality. We use bulk-sensitive soft-X-ray angle-resolved photoemission spectroscopy (SX-ARPES) to follow hybridization of the GeTe valence band with the Mn dopants. We observe a gradual opening of the Zeeman gap in the bulk Rashba bands around the Dirac point with increase of the Mn concentration, indicative of the ferromagnetic order, at persistent Rashba splitting. Furthermore, subtle details regarding the spin-orbit and magnetic order entanglement are deduced from spin-resolved ARPES measurements. We identify antiparallel orientation of the ferroelectric and ferromagnetic polarization, and altering of the Rashba-type spin helicity by magnetic switching. Our experimental results are supported by first-principles calculations of the electron and spin structure.

Energy metabolism of human LoVo colon carcinoma cells: correlation to drug resistance and influence of lonidamine.

The relationship between modification of energy metabolism and extent of drug resistance was investigated in two sublines (LoVoDX and LoVoDX10) from human LoVo colon carcinoma cells that exhibit different degrees of resistance to doxorubicin. Results indicated that the extent of alteration in energy metabolism strictly correlated with degree of resistance. In LoVoDX cells, only 14CO2 production was enhanced, whereas in the more resistant LoVoDX10 cells, both 14CO2 and aerobic lactate production were stimulated. The basal and glucose-supported efflux rate and the amount of drug extruded by LoVoDX10 cells were significantly higher than in the resistant LoVoDX cells. Because the expression of surface P-170 glycoprotein was similar in both cell lines, this phenomenon was attributed to increased efflux pump activity resulting from greater ATP availability. Inhibition of 14CO2 production, aerobic glycolysis, and clonogenic activity by lonidamine (LND) increased with enhancement of the energy metabolism. Moreover, LND, by affecting energy-yielding processes, reduced intracellular ATP content, lowered the energy supply to the ATP-driven efflux pump, and inhibited, almost completely, doxorubicin extrusion by resistant LoVo cells. These findings strongly suggest that LND, currently used in tumor therapy, reduces drug resistance by restoring the capacity to accumulate and retain drug of cells with the MDR phenotype that overexpress P-170.