Molecular subtypes of lung adenocarcinoma present distinct immune tumor microenvironments.

Overcoming resistance to immune checkpoint inhibitors is an important issue in patients with non-small-cell lung cancer (NSCLC). Transcriptome analysis shows that adenocarcinoma can be divided into three molecular subtypes: terminal respiratory unit (TRU), proximal proliferative (PP), and proximal inflammatory (PI), and squamous cell carcinoma (LUSQ) into four. However, the immunological characteristics of these subtypes are not fully understood. In this study, we investigated the immune landscape of NSCLC tissues in molecular subtypes using a multi-omics dataset, including tumor-infiltrating leukocytes (TILs) analyzed using flow cytometry, RNA sequences, whole exome sequences, metabolomic analysis, and clinicopathologic findings. In the PI subtype, the number of TILs increased and the immune response in the tumor microenvironment (TME) was activated, as indicated by high levels of tertiary lymphoid structures, and high cytotoxic marker levels. Patient prognosis was worse in the PP subtype than in other adenocarcinoma subtypes. Glucose transporter 1 (GLUT1) expression levels were upregulated and lactate accumulated in the TME of the PP subtype. This could lead to the formation of an immunosuppressive TME, including the inactivation of antigen-presenting cells. The TRU subtype had low biological malignancy and "cold" tumor-immune phenotypes. Squamous cell carcinoma (LUSQ) did not show distinct immunological characteristics in its respective subtypes. Elucidation of the immune characteristics of molecular subtypes could lead to the development of personalized immune therapy for lung cancer. Immune checkpoint inhibitors could be an effective treatment for the PI subtype. Glycolysis is a potential target for converting an immunosuppressive TME into an antitumorigenic TME in the PP subtype.

Identification of potential blood-based biomarkers for frailty by using an integrative approach.

INTRODUCTION: Although frailty is a geriatric syndrome that is associated with disability, hospitalization, and mortality, it can be reversible and preventable with the appropriate interventions. Additionally, as the current diagnostic criteria for frailty include only physical, psychological, cognitive and social measurements, there is a need for promising blood-based molecular biomarkers to aid in the diagnosis of frailty. METHODS: To identify candidate blood-based biomarkers that can enhance current diagnosis of frailty, we conducted a comprehensive analysis of clinical data, messenger RNA sequencing (RNA-seq), and aging-related factors using a total of 104 older adults aged 65-90 years (61 frail subjects and 43 robust subjects) in a cross-sectional case-control study. RESULTS: We identified two candidate biomarkers of frailty from the clinical data analysis, nine from the RNA-seq analysis, and six from the aging-related factors analysis. By using combinations of the candidate biomarkers and clinical information, we constructed risk-prediction models. The best models used combinations that included skeletal muscle mass index measured by dual-energy X-ray absorptiometry (adjusted p = 0.026), GDF15 (adjusted p = 1.46E-03), Adiponectin (adjusted p = 0.012), CXCL9 (adjusted p = 0.011), or Apelin (adjusted p = 0.020) as the biomarker. These models achieved a high area under the curve of 0.95 in an independent validation cohort (95% confidence interval: 0.79-0.97). Our risk prediction models showed significantly higher areas under the curve than did models constructed using only basic clinical information (Welch's t-test p < 0.001). CONCLUSION: All five biomarkers showed statistically significant correlations with components of the frailty diagnostic criteria. We discovered several potential biomarkers for the diagnosis of frailty. Further refinement may lead to their future clinical use.

Trans-ethnic genomic informed risk assessment for Alzheimer's disease: An International Hundred K+ Cohorts Consortium study.

BACKGROUND: As a collaboration model between the International HundredK+ Cohorts Consortium (IHCC) and the Davos Alzheimer's Collaborative (DAC), our aim was to develop a trans-ethnic genomic informed risk assessment (GIRA) algorithm for Alzheimer's disease (AD). METHODS: The GIRA model was created to include polygenic risk score calculated from the AD genome-wide association study loci, the apolipoprotein E haplotypes, and non-genetic covariates including age, sex, and the first three principal components of population substructure. RESULTS: We validated the performance of the GIRA model in different populations. The proteomic study in the participant sites identified proteins related to female infertility and autoimmune thyroiditis and associated with the risk scores of AD. CONCLUSIONS: As the initial effort by the IHCC to leverage existing large-scale datasets in a collaborative setting with DAC, we developed a trans-ethnic GIRA for AD with the potential of identifying individuals at high risk of developing AD for future clinical applications.

Tumor-infiltrating Leukocyte Profiling Defines Three Immune Subtypes of NSCLC with Distinct Signaling Pathways and Genetic Alterations.

Resistance to immune checkpoint blockade remains challenging in patients with non-small cell lung cancer (NSCLC). Tumor-infiltrating leukocyte (TIL) quantity, composition, and activation status profoundly influence responsiveness to cancer immunotherapy. This study examined the immune landscape in the NSCLC tumor microenvironment by analyzing TIL profiles of 281 fresh resected NSCLC tissues. Unsupervised clustering based on numbers and percentages of 30 TIL types classified adenocarcinoma (LUAD) and squamous cell carcinoma (LUSQ) into the cold, myeloid cell-dominant, and CD8+ T cell-dominant subtypes. These were significantly correlated with patient prognosis; the myeloid cell subtype had worse outcomes than the others. Integrated genomic and transcriptomic analyses, including RNA sequencing, whole-exome sequencing, T-cell receptor repertoire, and metabolomics of tumor tissue, revealed that immune reaction-related signaling pathways were inactivated, while the glycolysis and K-ras signaling pathways activated in LUAD and LUSQ myeloid cell subtypes. Cases with ALK and ROS1 fusion genes were enriched in the LUAD myeloid subtype, and the frequency of TERT copy-number variations was higher in LUSQ myeloid subtype than in the others. These classifications of NSCLC based on TIL status may be useful for developing personalized immune therapies for NSCLC. Significance: The precise TIL profiling classified NSCLC into novel three immune subtypes that correlates with patient outcome, identifying subtype-specific molecular pathways and genomic alterations that should play important roles in constructing subtype-specific immune tumor microenvironments. These classifications of NSCLC based on TIL status are useful for developing personalized immune therapies for NSCLC.

RNA-Sequencing Analysis Identification of Potential Biomarkers for Diagnosis of Sarcopenia.

Sarcopenia is a geriatric disease associated with increased mortality and disability. Early diagnosis and intervention are required to prevent it. This study investigated biomarkers for sarcopenia by using a combination of comprehensive clinical data and messenger RNA-sequencing (RNA-seq) analysis obtained from peripheral blood mononuclear cells. We enrolled a total of 114 older adults aged 66-94 years (52 sarcopenia diagnosed according to the Asian Working Group for Sarcopenia 2019 consensus and 62 normal older people). We used clinical data which were not included diagnosis criteria of sarcopenia, and stride length showed significance by logistic regression analysis (Bonferroni corrected p = .012, odds ratio = 0.14, 95% confidence interval [CI]: 0.05-0.40). RNA-seq analysis detected 6 differential expressed genes (FAR1, GNL2, HERC5, MRPL47, NUBP2, and S100A11). We also performed gene-set enrichment analysis and detected 2 functional modules (ie, hub genes, MYH9, and FLNA). By using any combination of the 9 candidates and basic information (age and sex), risk-prediction models were constructed. The best model by using a combination of stride length, HERC5, S100A11, and FLNA, achieved a high area under the curve (AUC) of 0.91 in a validation cohort (95% CI: 0.78-0.95). The quantitative PCR results of the 3 genes were consistent with the trend observed in the RNA-seq results. When BMI was added, the model achieved a high AUC of 0.95 (95% CI: 0.84-0.99). We have discovered potential biomarkers for the diagnosis of sarcopenia. Further refinement may lead to their future practical use in clinical use.

Identification of potential blood biomarkers for early diagnosis of Alzheimer's disease through immune landscape analysis.

Mild cognitive impairment (MCI) is a clinical precursor of Alzheimer's disease (AD). Recent genetic studies have reported on associations between AD risk genes and immunity. Here, we obtained samples and data from 317 AD, 432 MCI, and 107 cognitively normal (CN) subjects and investigated immune-cell type composition and immune clonal diversity of T-cell receptor (TRA, TRB, TRG, and TRD) and B-cell receptor (IGH, IGK, and IGL) repertoires through bulk RNA sequencing. We found the proportions of plasma cells, γδ T cells, neutrophils, and B cells were significantly different and the diversities of IGH, IGK, and TRA were significantly small with AD progression. We then identified a differentially expressed gene, WDR37, in terms of risk of MCI-to-AD conversion. Our prognosis prediction model using the potential blood-based biomarkers for early AD diagnosis, which combined two immune repertoires (IGK and TRA), WDR37, and clinical information, successfully classified MCI patients into two groups, low and high, in terms of risk of MCI-to-AD conversion (log-rank test P = 2.57e-3). It achieved a concordance index of 0.694 in a discovery cohort and of 0.643 in an independent validation cohort. We believe that further investigation, using larger sample sizes, will lead to practical clinical use in the near future.

Whole-genome sequencing reveals novel ethnicity-specific rare variants associated with Alzheimer's disease.

Alzheimer's disease (AD) is the most common multifactorial neurodegenerative disease among elderly people. Genome-wide association studies (GWAS) have been highly successful in identifying genetic risk factors. However, GWAS investigate common variants, which tend to have small effect sizes, and rare variants with potentially larger phenotypic effects have not been sufficiently investigated. Whole-genome sequencing (WGS) enables us to detect those rare variants. Here, we performed rare-variant association studies by using WGS data from 140 individuals with probable AD and 798 cognitively normal elder controls (CN), as well as single-nucleotide polymorphism genotyping data from an independent large Japanese AD cohort of 1604 AD and 1235 CN subjects. We identified two rare variants as candidates for AD association: a missense variant in OR51G1 (rs146006146, c.815 G > A, p.R272H) and a stop-gain variant in MLKL (rs763812068, c.142 C > T, p.Q48X). Subsequent in vitro functional analysis revealed that the MLKL stop-gain variant can contribute to increases not only in abnormal cells that should die by programmed cell death but do not, but also in the ratio of Aß42 to Aß40. We further detected AD candidate genes through gene-based association tests of rare variants; a network-based meta-analysis using these candidates identified four functionally important hub genes (NCOR2, PLEC, DMD, and NEDD4). Our findings will contribute to the understanding of AD and provide novel insights into its pathogenic mechanisms that can be used in future studies.

A functional variant of SHARPIN confers increased risk of late-onset Alzheimer's disease.

Late-onset Alzheimer's disease (LOAD) is the most common form of dementia, and its pathogenesis is multifactorial. We previously reported a rare functional variant of SHARPIN (rs572750141, NP_112236.3:p.Gly186Arg) that was significantly associated with LOAD. In addition, several recent studies have suggested the potential role of SHARPIN in AD pathogenesis. In this study, we sought to identify additional functional variants of SHARPIN in Japanese population. Six highly deleterious variants of SHARPIN, comprising four missense variants, one frameshift variant, and one stop-gain variant were detected from whole-genome sequencing data for 180 patients with LOAD and 184 with mild cognitive impairment. One of these candidate variants (rs77359862, NP_112236.3:p.Arg274Trp) was significantly associated with an increased risk of LOAD in 5043 LOAD cases and 11984 controls (P = 0.0016, odds ratio = 1.43). Furthermore, this variant SHARPIN showed aberrant cellular localization and reduced the activation of NF-κB, a central mediator of inflammatory and immune responses. Further investigation of the physiologic role of SHARPIN may reveal the mechanism of onset of LOAD.

Germ cell-specific expression of Venus by Tol2-mediated transgenesis in endangered endemic cyprinid Honmoroko (Gnathopogon caerulescens).

Fishes expressing a fluorescent protein in germ cells are useful to perform germ cell transfer experiments for conservation study. Nonetheless, no such fish has been generated in endangered endemic fishes. In this study, we tried to produce a fish expressing Venus fluorescent protein in germ cells using Honmoroko (Gnathopogon caerulescens), which is one of the threatened small cyprinid endemic to the ancient Lake Biwa in Japan. To achieve germ cell-specific expression of Venus, we used piwil1 (formally known as ziwi) promoter and Tol2 transposon system. Following the co-injection of the piwil1-Venus expression vector and the Tol2 transposase mRNA into fertilized eggs, presumptive transgenic fish were reared. At 7 months of post-fertilization, about 19% (10/52) of the examined larvae showed Venus fluorescence in their gonad specifically. Immunohistological staining and in vitro spermatogenesis using gonads of the juvenile founder fish revealed that Venus expression was detected in spermatogonia and spermatocyte in male, and oogonia and stage I and II oocytes in female. These results indicate that the Tol2 transposon and zebrafish piwil1 promoter enabled gene transfer and germ cell-specific expression of Venus in G. caerulescens. In addition, in vitro culture of juvenile spermatogonia enables the rapid validation of temporal expression of transgene during spermatogenesis.

Ethnic and trans-ethnic genome-wide association studies identify new loci influencing Japanese Alzheimer's disease risk.

Alzheimer's disease (AD) has no cure, but early detection and risk prediction could allow earlier intervention. Genetic risk factors may differ between ethnic populations. To discover novel susceptibility loci of AD in the Japanese population, we conducted a genome-wide association study (GWAS) with 3962 AD cases and 4074 controls. Out of 4,852,957 genetic markers that passed stringent quality control filters, 134 in nine loci, including APOE and SORL1, were convincingly associated with AD. Lead SNPs located in seven novel loci were genotyped in an independent Japanese AD case-control cohort. The novel locus FAM47E reached genome-wide significance in a meta-analysis of association results. This is the first report associating the FAM47E locus with AD in the Japanese population. A trans-ethnic meta-analysis combining the results of the Japanese data sets with summary statistics from stage 1 data of the International Genomics of Alzheimer's Project identified an additional novel susceptibility locus in OR2B2. Our data highlight the importance of performing GWAS in non-European populations.

Lower DNA methylation levels in CpG island shores of CR1, CLU, and PICALM in the blood of Japanese Alzheimer's disease patients.

The aim of the present study was to (1) investigate the relationship between late-onset Alzheimer's disease (AD) and DNA methylation levels in six of the top seven AD-associated genes identified through a meta-analysis of recent genome wide association studies, APOE, BIN1, PICALM, CR1, CLU, and ABCA7, in blood, and (2) examine its applicability to the diagnosis of AD. We examined methylation differences at CpG island shores in the six genes using Sanger sequencing, and one of two groups of 48 AD patients and 48 elderly controls was used for a test or replication analysis. We found that methylation levels in three out of the six genes, CR1, CLU, and PICALM, were significantly lower in AD subjects. The combination of CLU methylation levels and the APOE genotype classified AD patients with AUC = 0.84 and 0.80 in the test and replication analyses, respectively. Our study implicates methylation differences at the CpG island shores of AD-associated genes in the onset of AD and suggests their diagnostic value.

A rare functional variant of SHARPIN attenuates the inflammatory response and associates with increased risk of late-onset Alzheimer's disease.

BACKGROUND: Late-onset Alzheimer's disease (LOAD), the most common form of dementia, results from complicated interactions among multiple environmental and genetic factors. Despite recent advances in genetic analysis of LOAD, more than half of the heritability for the disease remains unclear. Although genetic studies in Caucasians found rare risk variants for LOAD with large effect sizes, these variants are hardly detectable in the Japanese population. METHODS: To identify rare variants possibly explaining part of the genetic architecture for LOAD in Japanese people, we performed whole-exome sequencing analyses of 202 LOAD individuals without the APOE ε4 risk allele, a major genetic factor for LOAD susceptibility. We also implemented in vitro functional analyses of the variant(s) to reveal possible functions associated with LOAD risk. RESULTS: Via step-by-step selection of whole-exome variants, we found seven candidate risk variants. We then conducted a case-control association study in a large Japanese cohort consisting of 4563 cases and 16,459 controls. We finally identified a rare nonsynonymous variant, rs572750141 (NM_030974.3:p.Gly186Arg), in SHARPIN that was potentially associated with increased risk of LOAD (corrected P = 8.05 × 10- 5, odds ratio = 6.1). The amino acid change in SHARPIN resulted in aberrant cellular localization of the variant protein and attenuated the activation of NF-κB, a central mediator of inflammatory and immune responses. CONCLUSIONS: Our work identified a rare functional SHARPIN variant as a previously unknown genetic risk factor for LOAD. The functional alteration in SHARPIN induced by the rare coding variant is associated with an attenuated inflammatory/immune response that may promote LOAD development.

Gic1 is a novel heterochromatin boundary protein in vivo.

In Saccharomyces cerevisiae, HMR/HML, telomeres and ribosomal DNA are heterochromatin-like regions in which gene transcription is prevented by the silent information regulator (Sir) complex. The Sir complex (Sir2, Sir3 and Sir4) can spread through chromatin from the silencer. Boundaries prevent Sir complex spreading, and we previously identified 55 boundary genes among all ~6,000 yeast genes. These boundary proteins can be distinguished into two types: those that activate transcription to prevent spreading of silencing, and those that prevent gene silencing by forming a boundary. We selected 44 transcription-independent boundary proteins from the 55 boundary genes by performing a one-hybrid assay and focused on GIC1 (GTPase interaction component 1). Gic1 is an effector of Cdc42, which belongs to the Rho family of small GTPases, and has not been reported to function in heterochromatin boundaries in vivo. We detected a novel boundary-forming activity of Gic1 at HMR-left and telomeric regions by conducting a chromatin immunoprecipitation assay with an anti-Sir3 antibody. We also found that Gic1 bound weakly to histones in two-hybrid analysis. Moreover, we performed domain analysis to identify domain(s) of Gic1 that are important for its boundary activity, and identified two minimum domains, which are located outside its Cdc42-binding domain.

Analysis of novel Sir3 binding regions in Saccharomyces cerevisiae.

In Saccharomyces cerevisiae, the HMR, HML, telomere and rDNA regions are silenced. Silencing at the rDNA region requires Sir2, and silencing at the HMR, HML and telomere regions requires binding of a protein complex, consisting of Sir2, Sir3 and Sir4, that mediates repression of gene expression. Here, several novel Sir3 binding domains, termed CN domains (Chromosomal Novel Sir3 binding region), were identified using chromatin immunoprecipitation (ChIP) on chip analysis of S. cerevisiae chromosomes. Furthermore, analysis of G1-arrested cells demonstrated that Sir3 binding was elevated in G1-arrested cells compared with logarithmically growing asynchronous cells, and that Sir3 binding varied with the cell cycle. In addition to 14 CN regions identified from analysis of logarithmically growing asynchronous cells (CN1-14), 11 CN regions were identified from G1-arrested cells (CN15-25). Gene expression at some CN regions did not differ between WT and sir3Δ strains. Sir3 at conventional heterochromatic regions is thought to be recruited to chromosomes by Sir2 and Sir4; however, in this study, Sir3 binding occurred at some CN regions even in sir2Δ and sir4Δ backgrounds. Taken together, our results suggest that Sir3 exhibits novel binding parameters and gene regulatory functions at the CN binding domains.