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.

Experimental colitis in IL-10-deficient mice ameliorates in the absence of PTPN22.

Interleukin (IL)-10 plays a key role in controlling intestinal inflammation. IL-10-deficient mice and patients with mutations in IL-10 or its receptor, IL-10R, show increased susceptibility to inflammatory bowel diseases (IBD). Protein tyrosine phosphatase, non-receptor type 22 (PTPN22) controls immune cell activation and the equilibrium between regulatory and effector T cells, playing an important role in controlling immune homoeostasis of the gut. Here, we examined the role of PTPN22 in intestinal inflammation of IL-10-deficient (IL-10-/- ) mice. We crossed IL-10-/- mice with PTPN22-/- mice to generate PTPN22-/- IL-10-/- double knock-out mice and induced colitis with dextran sodium sulphate (DSS). In line with previous reports, DSS-induced acute and chronic colitis was exacerbated in IL-10-/- mice compared to wild-type (WT) controls. However, PTPN22-/- IL-10-/- double knock-out mice developed milder disease compared to IL-10-/- mice. IL-17-promoting innate cytokines and T helper type 17 (Th17) cells were markedly increased in PTPN22-/- IL-10-/- mice, but did not provide a protctive function. CXCL1/KC was also increased in PTPN22-/- IL-10-/- mice, but therapeutic injection of CXCL1/KC in IL-10-/- mice did not ameliorate colitis. These results show that PTPN22 promotes intestinal inflammation in IL-10-deficient mice, suggesting that therapeutic targeting of PTPN22 might be beneficial in patients with IBD and mutations in IL-10 and IL-10R.

Effects of ambient air pressure on surface structures produced by ultrashort laser pulse irradiation.

We report an experimental analysis addressing striking effects of residual air ambient pressure, from atmospheric conditions (103 mbar) to high vacuum (10-4 mbar), on the surface structures induced on a silicon target by direct femtosecond laser irradiation. We observe an interesting direct impact of the ambient pressure on the period and depth of the generated ripples as well as on the formation of microgrooves. Moreover, a significant correlation is observed between the ripples' period and depth. The change of pressure is accompanied by a variation of the degree of nanoparticle coverage, which is eventually recognized as an important factor for the development of the final surface structures. These results shed light on the intriguing mechanisms underlying the formation of the various surface textures, also evidencing that the ambient pressure can act as an effective parameter to tailor some characteristic features of the processed surface.

Laser ablation of silicon induced by a femtosecond optical vortex beam.

We investigate laser ablation of crystalline silicon induced by a femtosecond optical vortex beam, addressing how beam properties can be obtained by analyzing the ablation crater. The morphology of the surface structures formed in the annular crater surface allows direct visualization of the beam polarization, while analysis of the crater size provides beam spot parameters. We also determine the diverse threshold fluences for the formation of various complex microstructures generated within the annular laser spot on the silicon sample. Our analysis indicates an incubation behavior of the threshold fluence as a function of the number of laser pulses, independent of the optical vortex polarization, in weak focusing conditions.

Optical response of Sr2RuO4 reveals universal fermi-liquid scaling and quasiparticles beyond Landau theory.

We report optical measurements demonstrating that the low-energy relaxation rate (1/τ) of the conduction electrons in Sr(2)RuO(4) obeys scaling relations for its frequency (ω) and temperature (T) dependence in accordance with Fermi-liquid theory. In the thermal relaxation regime, 1/τ ∝ (hω)(2)+(pπk(B)T)(2) with p = 2, and ω/T scaling applies. Many-body electronic structure calculations using dynamical mean-field theory confirm the low-energy Fermi-liquid scaling and provide quantitative understanding of the deviations from Fermi-liquid behavior at higher energy and temperature. The excess optical spectral weight in this regime provides evidence for strongly dispersing "resilient" quasiparticle excitations above the Fermi energy.

Guiding antiferromagnetic transitions in Ca[Formula: see text]RuO[Formula: see text].

Understanding and controlling the transition between antiferromagnetic states having different symmetry content with respect to time-inversion and space-group operations are fundamental challenges for the design of magnetic phases with topologically nontrivial character. Here, we consider a paradigmatic antiferromagnetic oxide insulator, Ca[Formula: see text]RuO[Formula: see text], with symmetrically distinct magnetic ground states and unveil a novel path to guide the transition between them. The magnetic changeover results from structural and orbital reconstruction at the transition metal site that in turn arise as a consequence of substitutional doping. By means of resonant X-ray diffraction we track the evolution of the structural, magnetic, and orbital degrees of freedom for Mn doped Ca[Formula: see text]RuO[Formula: see text] to demonstrate the mechanisms which drive the antiferromagnetic transition. While our analysis focuses on a specific case of substitution, we show that any perturbation that can impact in a similar way on the crystal structure, by reconstructing the induced spin-orbital exchange, is able to drive the antiferromagnetic reorganization.

Electron backscattering diffraction and X-ray diffraction studies of interface relationships in Sr3Ru2O7/Sr2RuO4 eutectic crystals.

Sr3Ru2O7/Sr2RuO4 eutectic system is investigated by electron backscattering diffraction (EBSD) and X-ray diffraction (XRD). The eutectic growth enables the solidification of the two phases in an ordered lamellar pattern extending along the growth direction, namely the b-axis direction. The eutectic material thus provides in the a-c plane two distinct interfaces having different microstructures with respect to the growth direction. Our analysis shows that, across the inplane c-axis direction (characterized by a poor lattice matching), the b-axis orientation is not constant at the individual interfaces, showing an orientation spread of about 5°. However, across the in-plane a-axis direction (characterized by a good lattice matching), the b-axis orientation does not change within a few tenths of degree (about 0.25°). Such information at nanoscale is also verified on a macroscopic level by standard XRD investigation.

Prognostic role of immunohistochemical overexpression of the p16 protein in women under the age of 35 and diagnosed with HSIL (CIN2) subjected to "cervix sparing" excision.

OBJECTIVE: To evaluate the role of immunohistochemical staining overexpression of p16 protein (p16 IHC) as a prognostic factor of persistence or recurrence of intraepithelial disease after excision procedure in young women diagnosed with HSIL (CIN2). PATIENTS AND METHODS: 62 women with a histological diagnosis of HSIL (CIN2) subjected to "cervix sparing" excisional procedure were included in this retrospective study. All had age less than or equal to 35 years, negative history of immunosuppression, available follow-up, and assessment of the resection margins state. Immunohistochemical staining for the p16 protein was evaluated on reviewed and confirmed HSIL (CIN2) histological specimens with negative resection margins. The post-treatment follow-up, including cytology, colposcopy, and histology, ranged from a minimum of 6 months to a maximum of 60 months. The persistence or recurrence of SIL during the follow-up period was based on histologic referral and defined as "the presence of SIL", "the presence of HSIL" and "progression to HSIL (CIN3)". RESULTS: 31/62 patients were positive for immunostaining (p16 IHC+), and 31/62 were negative (p16 IHC-). Persistence or recurrence after excision occurred more frequently within the p16 IHC+ than in p16 IHC- group, both as SIL (29% p16 IHC- vs. 32.3% p16 IHC+, p = 0.783) and HSIL (6.5% p16 IHC- vs. 12.9% p16 IHC+, p = 0.671). None of the patients in the p16 IHC- group showed progression to CIN3 for the entire observation period, whereas 9.7% of p16 IHC+ women progressed to CIN3 lesion (p = 0.042). The p16 IHC positivity showed a significant association with progression to CIN3 in 5 years of follow-up (p = 0.029) and with the presence of SIL after two years of follow-up (p = 0.031). The differences between the two groups increased after two years post-treatment: the p16 IHC- patients still had SIL only in 3.2% of cases and no longer had HSIL, while the p16 IHC+ women still showed SIL in 19.4% and HSIL in 6.5% of cases. The negative predictive value (NPV) of p16 IHC in predicting SIL's presence after treatment increased with the severity of the lesion (NPV for SIL 70.97%, for HSIL 93.55%, for CIN3 100%). CONCLUSIONS: The study suggests that young patients with p16 IHC- HSIL (CIN2) have a better post-excisional course of the cervical intraepithelial disease compared to p16 IHC+ women and that p16 IHC could have prognostic utility during the long-term follow-up, especially in forecasting progression to CIN3 in consideration of the high NPV (up to 100%). The efficacy of the adjuvant HPV vaccination in the management of HSIL (CIN2) p16+ young women is to be evaluated as part of the fertility-sparing treatment.

Unveiling unconventional magnetism at the surface of Sr2RuO4.

Materials with strongly correlated electrons often exhibit interesting physical properties. An example of these materials is the layered oxide perovskite Sr2RuO4, which has been intensively investigated due to its unusual properties. Whilst the debate on the symmetry of the superconducting state in Sr2RuO4 is still ongoing, a deeper understanding of the Sr2RuO4 normal state appears crucial as this is the background in which electron pairing occurs. Here, by using low-energy muon spin spectroscopy we discover the existence of surface magnetism in Sr2RuO4 in its normal state. We detect static weak dipolar fields yet manifesting at an onset temperature higher than 50 K. We ascribe this unconventional magnetism to orbital loop currents forming at the reconstructed Sr2RuO4 surface. Our observations set a reference for the discovery of the same magnetic phase in other materials and unveil an electronic ordering mechanism that can influence electron pairing with broken time reversal symmetry.

Structure, morphology and composition of natural junctions of Sr(2)RuO(4)-Sr(3)Ru(2)O(7) eutectic crystals.

A detailed study of morphological, compositional and structural aspects of Sr(2)RuO(4)-Sr(3)Ru(2)O(7) eutectic crystals is reported. The stoichiometry of the phases that compose the crystals and how they are arranged in the crystals are studied. Understanding the behavior at the Sr(2)RuO(4)-Sr(3)Ru(2)O(7) interface represents a necessary prerequisite for the analysis of the experimental results on the transport properties recently reported in the literature.

Structural, electrical and magnetic characterization of artificial ferromagnetic/superconducting (La(0.7)Ca(0.3)MnO(3)/YBa(2)Cu(3)O(7-x)) heterostructures.

The fabrication and characterization of superconducting and ferromagnetic heterostructures is an open field due to the fundamental interest in the physics of the coexistence of these two competing orders and their possible applications in the spintronics industry. In this paper we present structural, electrical and magnetic characterization for the single La(0.7)Ca(0.3)MnO(3) (LCMO) thin layer, La(0.7)Ca(0.3)MnO(3)/YBa(2)Cu(3)O(7-x) (LCMO/YBCO) bilayers and the LCMO/YBCO/LCMO trilayers. In particular, we show a detailed magnetic characterization of the LCMO thin films by means of low temperature magnetic force microscopy. We discuss the different dynamics of the magnetic domains observed, depending on the substrate induced strain and on the film thickness.

Microcolposcopy in the diagnostic evaluation of abnormal cervical cytology: when and why to do it.

Microcolposcopy is an in vivo cytological examination of the uterine cervix allowing the localization of exoendocervical precancerous lesions. The aim of this study was to assess the diagnostic reliability of microcolposcopy by means of correlation with histology, colposcopy and Pap test results. For the study, 256 patients with abnormal Pap test results were selected and subjected to colposcopy and microcolposcopy with the aim of evaluating the presence of any intraepithelial lesions. One hundred and nine of these patients were subjected to a biopsy. Colposcopy, histology and cytology results were compared with those obtained by microcolposcopy. In low-grade squamous intraepithelial lesion (LSIL) cytology cases, the percentage agreement on lesion grade between Pap test and microcolposcopy results was 74%, while in high-grade squamous intraepithelial lesion (HSIL) cytology cases, it was equal to 80%. The comparison between colposcopy and microcolposcopy showed a level of agreement of 72% for lower grades and 68% for higher grades. Finally, histology was in agreement with microcolposcopy in 73% of cervical intraepithelial grade 1 neoplasia (CIN 1) cases and reached 71% for CIN 2-3. Microcolposcopy proved to be accurate with regard to the diagnosis of lesion grade, and showed to be definitive in patients where cytology was positive for HPV infection and colposcopy was not able to identify any lesions.

Severe macroglossia after posterior fossa and craniofacial surgery in children.

Massive swelling of the tongue can occur after posterior fossa and craniofacial surgery. Several hypotheses have been proposed to explain the occurrence of such severe postoperative macroglossia, but this phenomenon is still poorly understood. Severe postoperative macroglossia can be a life-threatening condition due to upper airway obstruction. Three cases of severe postoperative macroglossia that occurred after cervical spine, craniofacial, and posterior fossa surgical procedures are reported here. These cases required specialized maxillofacial management and a prolonged stay in the intensive care unit. Causal factors involved in this condition are reported, in order to highlight appropriate prevention and treatment options adapted to the management of paediatric patients. An overview of the current literature on severe postoperative macroglossia in paediatric populations is also provided.

Antifungal activity of diketopiperazines extracted from Alternaria alternata against Plasmopara viticola: an ultrastructural study.

Three dipeptides, belonging to the family of diketopiperazines (DKPs), were extracted from broth culture of the grapevine endophyte Alternaria alternata, and were tested against Plasmopara viticola on leaves of grapevine plants grown in greenhouse. DKPs, used at different concentrations (10(-3), 10(-4), 10(-5) and 10(-6)M) both singularly and in mixtures, demonstrated real effectiveness in inhibiting P. viticola sporulation when applied 2 or 24h after pathogen inoculation. Moreover, no necrotic lesions or other phytotoxicity symptoms were observed on DKP-treated grapevine leaf tissues. Ultrastructural analysis performed on grapevine leaf tissues revealed that the DKPs used singularly and in mixture, at above reported concentrations, did not cause leaf tissue damages. By contrast, hyphae of P. viticola exhibited marked structural changes, similar to those induced by the endophyte A. alternata. This demonstrates the involvement of these metabolites in the relationship of P. viticola and the endophyte. Further experimental trials will be carried out in the next future in order to test the effectiveness of these molecules also under field conditions, and to better understand the mechanism of action involved in the pathogen inhibition.

Molecular genetics of prostate cancer: clinical translational opportunities.

Prostate cancer (PC) development reflects a complex sequence of biologic and molecular events. Several inheritable and somatic genetic changes have been identified. The knowledge of the molecular basis of PC can improve our understanding of the causes of this common cancer and provide information on prognosis and treatment. To date, however, no molecular studies have yet yielded consistent information that is ready to be incorporated into clinical practice. We reviewed the current literature on the molecular biology of prostate cancer and analyzed different potential tumor markers according to the classical concepts of oncogenes, suppressor genes, and the more modern concepts of genes involved in detoxification or inflammatory pathways of cancer progression. This review aims to identify trends in PC research and suggests potential clinical applications for diagnosis, prognosis, prevention and treatment.

Hallmarks of Hunds coupling in the Mott insulator Ca2RuO4.

A paradigmatic case of multi-band Mott physics including spin-orbit and Hund's coupling is realized in Ca2RuO4. Progress in understanding the nature of this Mott insulating phase has been impeded by the lack of knowledge about the low-energy electronic structure. Here we provide-using angle-resolved photoemission electron spectroscopy-the band structure of the paramagnetic insulating phase of Ca2RuO4 and show how it features several distinct energy scales. Comparison to a simple analysis of atomic multiplets provides a quantitative estimate of the Hund's coupling J=0.4 eV. Furthermore, the experimental spectra are in good agreement with electronic structure calculations performed with Dynamical Mean-Field Theory. The crystal field stabilization of the dxy orbital due to c-axis contraction is shown to be essential to explain the insulating phase. These results underscore the importance of multi-band physics, Coulomb interaction and Hund's coupling that together generate the Mott insulating state of Ca2RuO4.

Inhibition of Sporulation and Ultrastructural Alterations of Grapevine Downy Mildew by the Endophytic Fungus Alternaria alternata.

ABSTRACT One hundred twenty-six endophytic microorganisms isolated from grapevine leaves showing anomalous symptoms of downy mildew were tested on grapevine leaf disks as biocontrol agents against Plasmopara viticola. Among the 126 microorganisms, only five fungal isolates completely inhibited the sporulation of P. viticola; all of them were identified as Alternaria alternata. Ultrastructural analyses were carried out by transmission electron microscopy to observe cellular interactions between P. viticola and A. alternata in the grapevine leaf tissue. Cytological observations indicated that, even without close contact with A. alternata, the P. viticola mycelium showed severe ultrastructural alterations, such as the presence of enlarged vacuoles or vacuoles containing electron-dense precipitates. Haustoria appeared necrotic and irregularly shaped or were enclosed in callose-like substances. Therefore, a toxic action of A. alternata against P. viticola was hypothesized. To examine the production of toxic low-molecular-weight metabolites by A. alternata, we analyzed the fungal liquid culture by thin layer chromatography and proton magnetic resonance spectroscopy. The main low-molecular-weight metabolites produced by the endophyte were three diketopiperazines: cyclo(l-phenylalanine-trans-4-hydroxy-l-proline), cyclo(l-leucine-trans-4-hydroxy-l-proline), and cyclo(l-alanine-trans-4-hydroxy-l-proline). When applied at different concentrations to both grapevine leaf disks and greenhouse plants, a mixture of the three diketopiperazines was very efficacious in limiting P. viticola sporulation.

Molecular genetics of bladder cancer: targets for diagnosis and therapy.

Transitional cell carcinoma of the bladder is a common tumor. While most patients presenting superficial disease can be expected to do well following treatment, still many patients will return to our office with muscle invasive and metastatic disease. Survival in advanced bladder cancer is less than 50%. Tumors of similar histologic grade and stage have variable behavior, suggesting that genetic alterations must be present to explain the diverse behavior of bladder cancer. It is hoped that through the study of the subtle genetic alterations in bladder cancer, important prognostic and therapeutic targets can be exploited. Many new diagnostic tests and gene therapy approaches rely on the identification and targeting of these unique genetic alterations. A review of literature published on the molecular genetics of bladder cancer from 1970 to the present was conducted. A variety of molecular genetic alterations have been identified in bladder cancer. Oncogenes (H-ras, erbB-2, EGFR, MDM2, C-MYC, CCND1), tumor suppressor genes (p53, Rb, p21, p27/KIP1, p16, PTEN, STK15, FHIT, FEZ1/LZTS1, bc10), telomerase, and methylation have all been studied in bladder cancer. Several have proven to be potentially useful clinical targets in the prognosis and therapy of bladder cancer such as staining for p53 and gene therapy strategies such as p53 and fez1. Clinical trials targeting HER2/neu and the EGFR pathways are underway. The UroVysion bladder cancer assay relies on FISH to detect genetic alterations in this disease. Continuing identification of the molecular genetic alterations in bladder cancer will enhance future diagnostic and therapeutic approaches to bladder cancer. Capitalizing on these alterations will allow early detection, providing important prognostic information and unique targets for gene therapy and other therapeutic approaches.