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Background Genome-wide landscape of alternative promoter use remains unknown. We determined expression profiles of promoters in 26 lung adenocarcinoma cell lines using the transcriptional start site-sequencing data and proposed an index ‘canonical promoter usage’ to quantify the diversity of alternative promoter usage. Methods Transcriptional start site-sequencing and other datasets were obtained from the DataBase of Transcriptional Start Sites. Transcriptional start site-sequencing read clusters were mapped onto RefGene to determine the promoters. Commonly used promoters were designated as canonical promoters. The sequence logos, CpG islands, DNA methylation and histone modifications of canonical and non-canonical promoters were examined. Canonical promoter usage was calculated by dividing ‘read counts of a canonical promoter’ by ‘read counts of all the units of promoters’ on each gene. The expressed genes were subjected to hierarchical clustering according to their canonical promoter usage. Results Among 104 455 promoters for 14 297 genes, 8659 canonical and 68 197 non-canonical promoters were identified. Corresponding to higher expression, canonical promoters showed core promoter sequences, higher CpG island positivity, less DNA methylation and higher transcription-promoting histone modifications. Gene ontology enrichment analysis revealed that the clusters with lower canonical promoter usage were related to signalling pathways, whereas clusters of tightly regulated genes with higher canonical promoter usage were related to housekeeping genes. Conclusion Canonical promoters were regulated by conventional transcriptional machinery, while non-canonical promoters would be targets of ‘leaky’ expression. Further investigation is warranted to analyse the correlation between alternative promoter usage and biological characteristics contributing to carcinogenesis.

Background Irritable bowel syndrome (IBS) is a disorder of gut–brain interaction, including dysregulation of the hypothalamic–pituitary–adrenal axis with salivary cortisol changes. However, the role of gastrointestinal microbiota during IBS symptom exacerbation remains unclear. We tested the hypothesis that the microbial species, gene transcripts, and chemical composition of fecal and oral samples are altered during the exacerbation of IBS symptoms. Methods Fecal, salivary, and dental plaque samples were collected at baseline from 43 men with IBS with diarrhea (IBS-D) and 40 healthy control (HC) men. Samples in the IBS-D patients were also collected during symptom exacerbation. The composition of the fecal microbiota was determined by analyzing the 16S rRNA gene, RNA-based metatranscriptome, and metabolites in samples from HC and IBS patients with and without symptom exacerbation. Oral samples were also analyzed using omics approaches. Results The fecal microbiota during IBS symptom exacerbation exhibited significant differences in the phylogenic pattern and short-chain fatty acid compared with fecal samples during defecation when symptoms were not exacerbated. Although there were no significant differences in the phylogenic pattern of fecal microbiota abundance between HCs and IBS-D patients, significant differences were detected in the expression patterns of bacterial transcriptomes related to butyrate production and neuroendocrine hormones, including tryptophan-serotonin-melatonin synthesis and glutamine/GABA. The composition of plaque microbiota was different between HC and IBS-D patients during normal defecation. Conclusions Our findings suggest that colonic host-microbial interactions are altered in IBS-D patients during exacerbation of symptoms. There were no overlaps between feces and oral microbiomes.

Background Combination therapy based on radiotherapy and immune checkpoint inhibitors (ICIs) was recently reported as effective for various cancers. The radiation-induced immune response (RIIR) is an essential feature in ICI-combined radiotherapy; however, the effects of drugs used concomitantly with RIIR remain unclear. We screened for drugs that can modify RIIR to understand the mutual relationship between radiotherapy and combined drugs in ICI-combined radiotherapy. Methods We established a high-throughput system with reporter gene assays for evaluating RIIR, focusing on factors acting downstream of the STING-IRF pathway, which can stimulate cancer cells, T cells, and dendritic cells. We further quantified the effects of 2595 drugs, including those approved by the Food and Drug Administration, on RIIR in vitro. Results The reporter assay results correlated well with the expression of immune response proteins such as programmed death-ligand 1. This high-throughput system enabled the identification of drugs including cytotoxic agents, molecular-targeted agents, and other agents that activate or suppress RIIR. Conclusions Our study provides an encyclopedic catalogue of clinically approved drugs based on their effect on RIIR. In ICIs combined radiotherapy, activation of STING-IFN may improve the therapeutic effect and our result could form a biological basis for further clinical trials combining radiotherapy with ICIs.

Table S2. Nucleotide sequences of the primers and PCR conditions

Purpose:The epithelial-to-mesenchymal transition, the major process by which some cancer cells convert from an epithelial phenotype to a mesenchymal one, has been suggested to drive chemo-resistance and/or metastasis in patients with cancer. However, only a few studies have demonstrated the presence of CD45/CD326 doubly-positive cells (CD45/CD326 DPC) in cancer. We deployed a combination of cell surface markers to elucidate the phenotypic heterogeneity in non–small cell lung cancer (NSCLC) cells and identified a new subpopulation that is doubly-positive for epithelial and non–epithelial cell-surface markers in both NSCLC cells and patients' malignant pleural effusions.Experimental Design:We procured a total of 39 patients' samples, solid fresh lung cancer tissues from 21 patients and malignant pleural effusion samples from 18 others, and used FACS and fluorescence microscopy to check their surface markers. We also examined the EGFR mutations in patients with known acquired EGFR mutations.Results:Our data revealed that 0.4% to 17.9% of the solid tumor tissue cells and a higher percentage of malignant pleural effusion cells harbored CD45/CD326 DPC expressing both epithelial and nonepithelial surface markers. We selected 3 EGFR mutation patients and genetically confirmed that the newly identified cell population really originated from cancer cells. We also found that higher proportions of CD45/CD326 DPC are significantly associated with poor prognosis.Conclusions:In conclusion, varying percentages of CD45/CD326 DPC exist in both solid cancer tissue and malignant pleural effusion in patients with NSCLC. This CD45/CD326 doubly-positive subpopulation can be an important key to clinical management of patients with NSCLC.

Supplementary Figure S1. Genetic alterations of TP53 (exons 5 through 9) and KRAS (exon 2 containing codons 12 and 13) in Cases #1, 6, and 9 were Sanger sequenced; both strands were analyzed.

PURPOSE Genomic profiling programs have been implemented to apply next-generation sequencing (NGS) for facilitating trial enrollment. SCRUM-Japan GI-SCREEN is a large-scale genomic profiling program in advanced gastrointestinal cancers using a validated genomic assay with the goal of facilitating enrollment in targeted clinical trials, generating real-world data, and performing clinicogenomic analysis for biomarker discovery. PATIENTS AND METHODS Genotyping of tumor tissue samples from 5,743 patients with advanced gastrointestinal cancers enrolled in GI-SCREEN was centrally performed with NGS. Patients were enrolled in matched trials of targeted agents affiliated with GI-SCREEN on the basis of genotyping results. RESULTS A total of 11 gastrointestinal cancers were included, with colorectal cancer being the most common. The median age ranged from 59 to 70.5 years across cancer types. Patients enrolled after initiation of first-line treatment had significantly longer overall survival (OS) than that before treatment initiation with a median survival time difference of 8.9 months and a hazard ratio (HR) ranging from 0.25 to 0.73 across cancer types, demonstrating an immortal time bias. One hundred and forty-nine patients received matched therapies in clinical trials on the basis of their identified alterations. Among patients with colorectal cancer harboring actionable alterations, the median OS was significantly longer in patients who received matched therapies in trials than in those who did not (HR, 0.52; 95% CI, 0.26 to 1.01; P = .049). Cancer-specific pathway alterations were significantly associated with shorter survival and related to primary resistance to matched trial therapies. CONCLUSION Our genomic profiling program led to patient enrollment in targeted clinical trials and improved survival of patients with colorectal cancer who received matched therapies in clinical trials. To avoid immortal time bias, precautions are needed when using data from patients who have undergone NGS testing after initiation of the evaluated treatment line.

Radiotherapy (RT) is an effective treatment option for cancer; however, its efficacy remains less than optimal in locally advanced cancer. Immune checkpoint inhibitor-based therapy, including the administration of anti-PD-L1 antibodies, is a promising approach that works synergistically with RT. Proton beam therapy and carbon-ion therapy are common options for patients with cancer. Proton and carbon ions are reported to induce an immune reaction in cancer cells; however, the underlying mechanisms remain unclear. Here, we aimed to compare the immune responses after irradiation (IR) with X-ray, protons, and carbon ions in an oesophageal cancer cell line and the underlying mechanisms. An oesophageal cancer cell line, KYSE450, was irradiated with 1 fraction/15 GyE (Gy equivalent) of X-ray, proton, or carbon-ion beams, and then, the cells were harvested for RNA sequencing and gene enrichment analysis. We also knocked out STING and STAT1 in the quest for mechanistic insights. RNA sequencing data revealed that gene expression signatures and biological processes were different in KYSE450 irradiated with X-ray, proton, and carbon-ion beams 6–24 h after IR. However, after 3 days, a common gene expression signature was detected, associated with biological pathways involved in innate immune responses. Gene knock-out experiments revealed that the STING-STAT1 axis underlies the immune reactions after IR. X-Ray, proton, and carbon-ion IRs induced similar immune responses, regulated by the STING-STAT1 axis.

Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by inflammation of various organs such as skin, kidneys, bones, and brain and the presence of autoantibodies. Although the cause of SLE is not completely understood, environmental factors, genetic susceptibility, hormone factors, and environmental factors are thought to play essential roles in the pathogenesis of SLE. Among environmental factors, the microbiota are linked to the development of different autoimmune diseases. The microbiota in the nasal cavity and gut are involved in SLE development, but the influence of skin microbiota is still unclear. Here, we demonstrated that epithelial cell–specific IκBζ-deficient ( Nfkbiz ΔK5 ) mice showed spontaneous skin inflammation with increased abundance of Staphylococcus aureus on the skin. When S. aureus was epicutaneously applied on Nfkbiz ΔK5 mice, Nfkbiz ΔK5 mice developed SLE-associated autoantibodies, anti-dsDNA antibodies, anti-Sm antibodies, and glomerulonephritis with IgG deposition. Epicutaneous S. aureus application significantly increased staphylococcal colonization on the skin of Nfkbiz ΔK5 mice with reduced expression of several antimicrobial peptides in the skin. This staphylococcal skin colonization promoted caspase-mediated keratinocyte apoptosis and neutrophil activation, inducing the interleukin-23 (IL-23)/IL-17 immune response by activating dendritic cells and T cells. Furthermore, the subcutaneous administration of anti–IL-23p19 and anti–IL-17A antibodies alleviated the systemic autoimmune response. Together, these findings underscore epithelial-immune cross-talk disturbances caused by skin dysbiosis as an essential mediator inducing autoimmune diseases.

Gastric mesenchymal tumors are relatively rare, and their molecular pathogeneses are poorly understood, except for gastrointestinal stromal tumor, desmoid, and inflammatory myofibroblastic tumors. We report a case of a gastric mesenchymal tumor with prominent smooth muscle cell differentiation and an echinoderm microtubule-associated protein-like 4-anaplastic lymphoma kinase (EML4-ALK) fusion. On gross section, the tumor was 26 mm at the largest diameter, well-circumscribed, and located in the submucosal and muscular layers of the stomach wall. Histologically, the tumor comprised intersecting fascicles of spindle cells, non-atypical nuclei, and highly eosinophilic cytoplasm. Myxoid changes were observed focally, but inflammatory infiltrates were only evident in limited areas. Immunochemical staining revealed that the tumor was positive for α-smooth muscle actin and desmin. Diffuse positive staining for h-caldesmon was observed throughout the tumor, which suggested smooth muscle cell differentiation. Intracytoplasmic staining for ALK protein was also observed, and fluorescence in situ hybridization using ALK break-apart probes showed split chromosomal signals. RNA-sequencing analysis identified EML4-ALK fusion transcripts. We concluded that the tumor was a gastric mesenchymal tumor with smooth muscle differentiation based on its distinct differential smooth muscle properties, such as highly eosinophilic cytoplasm and diffuse expression of h-caldesmon. Furthermore, activated ALK may underly the tumor's pathogenesis.

Background and purpose Ionising radiation causes mutations in the genomes of tumour cells and serves as a potent treatment for cancer. However, the mutation signatures in the cancer genome following ionising radiation have not been documented. Materials and methods We established an in vitro experimental system to analyse the presence of de novo mutations in the cancer genome of irradiated (60 Gy/20 fr/4 weeks) oesophageal cancer cell lines. Subsequently, we performed whole-genome, chromatin immunoprecipitation, and RNA sequencing using untreated and irradiated samples to assess the damage to the genome caused by radiation and understand the underlying mechanism. Results The irradiated cancer cells exhibited hotspots for the de novo 8502–12966 single nucleotide variants and 954–1,331 indels on the chromosome. These single nucleotide variants primarily originated from double-stranded break repair errors, as determined using mutation signature analysis. The hotspots partially overlapped with the sites of H3K9 trimethylation, which are regions characterised by a weak capacity for double-stranded break repair. Conclusion This study highlights the signature and underlying mechanism of radiation on the cancer genome.

Human lymphoblastoid cell lines (LCLs) are valuable for the functional analyses of diseases. We have established more than 4200 LCLs as one of the resources of an integrated biobank. While oxidative and inflammatory stresses play critical roles in the onset and progression of various diseases, the responsiveness of LCLs, especially that of biobank-made LCLs, to these stresses has not been established. To address how LCLs respond to these stresses, in this study, we performed RNA sequencing of eleven human LCLs that were treated with an electrophile, diethyl maleate (DEM) and/or an inflammatory mediator, lipopolysaccharide (LPS). We found that over two thousand genes, including those regulated by a master regulator of the electrophilic/oxidative stress response, NRF2, were upregulated in LCLs treated with DEM, while approximately three hundred genes, including inflammation-related genes, were upregulated in LPS-treated LCLs. Of the LPS-induced genes, a subset of proinflammatory genes was repressed by DEM, supporting the notion that DEM suppresses the expression of proinflammatory genes through NRF2 activation. Conversely, a part of DEM-induced gene was repressed by LPS, suggesting reciprocal interference between electrophilic and inflammatory stress-mediated pathways. These data clearly demonstrate that LCLs maintain, by and large, responsive pathways against oxidative and inflammatory stresses and further endorse the usefulness of the LCL supply from the biobank.

Mutational signatures associated with tobacco smoking (mutational smoking signatures: SS) are characterized mainly by C A mutations. The aim of this study was to characterize the association between the tumor immune microenvironment and the SS in lung adenocarcinoma.Lung adenocarcinomas surgically resected from 96 patients, for which whole exome sequencing data was available, were included in the study. We extracted the SS from whole exome sequencing data, calculated the weights of SS using deconstructSigs, and compared the clinicopathological features of SS positive (SS+) and negative (SS-) adenocarcinomas. We selected 18 tumor pairs from SS + and SS- adenocarcinomas (sex, EGFR mutation, and tumor size-matched) and examined the expression of five immune markers (CD20, CD8, FOXP3, CD204, and PD-L1) by immunohistochemistry.Of 96 specimens, there were 33 (34 %) SS + adenocarcinoma tumors. The smoking index significantly correlated with the weight of the SS (R = 0.43). Between SS + and SS- tumors, there was no significant difference in clinicopathological factors excluding smoking history. Immunohistochemistry revealed that the number of FOXP3 + T cells in SS + adenocarcinomas was significantly higher than that in the SS- adenocarcinomas (median number 58 vs. 36, p 0.01). Also, the number of CD20 + B cells in SS + adenocarcinomas was significantly higher than that in the SS- adenocarcinomas (median number 77 vs. 29, p 0.01); however; these phenomena could not be confirmed when stratified by smoking history.In lung adenocarcinoma, SS is associated with an immunosuppressive tumor microenvironment.

Purpose Activated Notch receptor signaling has been implicated in tumor growth and progression in colorectal cancer (CRC). However, the pathogenic relevance of NOTCH gene alterations remains unclear. The aim of this study was to clarify mutational landscapes and assess their clinical significance in patients with metastatic CRC.Methods Pre-chemotherapy tumor tissues obtained from 1154 metastatic CRC patients in the Nationwide Cancer Genome Screening Project in Japan between April 2017 and March 2019 were studied using the Oncomine Comprehensive Assay.Results The frequencies of NOTCH1, NOTCH2, and NOTCH3 nonsynonymous sequence variants were 11.5%, 4.4%, and 10.4%, respectively. The majority of variants were missense of unknown significance that were distributed across all domains of all three NOTCH genes. The gain-of-function mutations in NOTCH reported in multiple malignancies were not identified. The NOTCH amplification rate was less than 1%. No NOTCH fusions were detected. In patients who were registered before, or within one year of, first-line chemotherapy, overall survival for 51 patients with only NOTCH3 variants was significantly longer than for 540 patients with no NOTCH variants (median, 40.2 months vs 27.7 months; P = 0.04). Multivariate analysis revealed that variant NOTCH3 was an independent prognostic factor for increased survival (hazard ratio 0.61, 95% confidence interval, 0.39-0.94; P = 0.03) besides poor prognostic factors associated with mutant TP53, KRAS, and BRAF, as well as amplified MYC.Conclusion NOTCH genes are unlikely to harbor driver mutations and amplifications in patients with metastatic CRC. NOTCH3 variants should be further investigated as a favorable prognostic marker.

213 Background: Preclinical and clinical data from various types of cancer show that the gut microbiome can affect the outcome of treatments with immune checkpoint inhibitors or cytotoxic chemotherapies. However, the relationship between the microbiome and treatment outcomes in prostate cancer has remained unclear. Here, we present a comprehensive analysis of the relationship between the gut microbiome and treatment outcomes from a nationwide genome screening project (MONSTAR-SCREEN 1) in patients with metastatic castration-sensitive and -resistant prostate cancer (mCSPC and mCRPC). Methods: We performed 16S rRNA gene sequencing of fecal DNA from 73 mCSPC and 83 mCRPC patients before treatment. The microbiome data were compared using the Wilcoxon-Mann-Whitney test or Fisher’s exact test. Survival status and therapeutic efficacy of ADT based treatment were prospectively collected and estimated using the Kaplan-Meier method, and the log-rank test was used to compare survival in different strata. Results: Fecal samples from mCRPC had more Klebsiella and Enterobacteriaceae than those from mCSPC, whereas mCSPC had more Akkermansia and Bifidobacterium compared to mCRPC. Prior and concurrent usage of anti-biotics did not affect diversity of the amplicon sequence variants (ASVs), KEGG orthology and metabolism pathway representation. Anti-biotics also did not influence time to treatment failure (TTF) in mCRPC (HR:0.81, 95%CI:0.36–1.78) and mCSPC (HR:1.35, 95%CI:0.55–3.34). mCRPC had more diverse KEGG orthology compared to mCSPC ( p=0.0009), however, no statistical differences were observed in the ASV ( p=0.77) and metabolism pathway representation ( p=0.35) between mCRPC and mCSPC. High ASV tended to be associated with longer TTF rather than low ASV in both mCRPC (Adjusted HR:0.63, 95%CI:0.34–1.10) and mCSPC (Adjusted HR:0.66, 95%CI:0.31–1.40). mCRPC patients with high AVS also showed significant longer PSA-progression free survival than patients with low ASV (HR:053, 95%CI:0.28–0.99). Conclusions: This investigation revealed significant differences in the microbiome status of mCRPC compared to mCSPC. These differences and diversity might influence the outcomes of ADT based treatment in prostate cancer.

11 Background: The PARADIGM trial showed longer median overall survival (OS) with first-line mFOLFOX6 plus panitumumab (PAN) vs bevacizumab (BEV) in patients (pts) with RAS wild type (WT) and left-sided metastatic colorectal cancer (mCRC) (37.9 vs 34.3 months, respectively; hazard ratio [HR], 0.82; P=0.031) and similar OS in right-sided pts (HR 1.09; Yoshino T, et al. ASCO 2022 LBA1). We evaluated the usefulness of negative hyperselection by gene alterations in ctDNA related to primary resistance to anti-EGFR therapy. Methods: Baseline plasma ctDNA (>10 ng/mL and >10 nM DNA) from pts enrolled in the biomarker study (NCT02394834) was assessed using a custom panel (PlasmaSELECT-R 91, PGDx). Preplanned gene alterations for hyperselection included KRAS, NRAS, PTEN, and extracellular domain EGFR mutations, HER2 and MET amplifications, and ALK, RET, or NTRK1 fusions. Results: Among 802 pts in the full analysis set, 733 (91%) had evaluable pretreatment samples for ctDNA analysis. Of these pts, 204 (28%) had at least 1 gene alteration, including KRAS/NRAS (8%), BRAF V600E (11%), PTEN (5%), EGFR (3%), HER2 (5%), MET (1%), and fusions (1%). In 529 (72%) hyperselected pts without any gene alterations, OS tended to be longer with PAN vs BEV regardless of primary sidedness; the OS HRs ranged from 0.76 to 0.82, and the median OS gains ranged from 6.6 to 8.0 months (Table). Meanwhile, OS was similar or inferior with PAN vs BEV irrespective of the primary sidedness in pts with any of these gene alterations (Table). Conclusions: Negative hyperselection using ctDNA rather than tumor sidedness may identify appropriate pts for first-line PAN over BEV. These results warrant further validation in additional cohorts. Clinical trial information: NCT02394834 . [Table: see text]

733 Background: MONSTAR-SCREEN prospectively assessed the gut microbiota of 2000 patients (pts) with advanced solid cancer at 31 Japanese institutions. Here, we report the interim analysis results of the correlation between gut microbiota and the efficacy of first-line chemotherapy in pts with unresectable pancreatic cancer (PC) and bile tract cancer (BTC). Methods: We analyzed unresectable 205 PC and 83 BTC pts with clinical data as data cut-off on April 30, 2021, and 16S ribosome analysis results of stool samples. Among PC pts, 133 and 40 were treated with GnP (gemcitabine (GEM) plus nab-paclitaxel) and FOLFIRINOX (5-fluorouracil, leucovorin, irinotecan, and oxaliplatin), respectively. Among BTC pts, 32 and 33 were treated with GC (GEM plus cisplatin) and GCS (GEM, cisplatin, plus S-1), respectively. Pts with an above-median overall survival (OS) were defined as long-term survivors (LTS), and those with below-median OS and OS events were defined as short-term survivors (STS). We compared the alpha diversity index (ADI) of LTS and STS, and identified potential microbial biomarkers of LTS, STS, responders, and non-responders to first-line chemotherapy according to RECIST version 1.1, by linear discriminant analysis effect size (LEfSe). In each cohort, the top three microbiota with linear discriminant analysis scores of ≥ 3.0 were considered significant. Results: The median OS of PC and BTC pts was 13.5 and 15.9 months, respectively. In PC pts, no significant difference was noted in the ADI between LTS (n=20) and STS (n=40). In BTC pts, the ADI of LTS (n=10) was significantly higher than that of STS (n=20) in the Shannon index. LEfSe analysis in PC pts revealed that Burkholderiales and Sutterellaceae were predominant in LTS, while Lactobacillaceae was, in STS. In PC pts, Brachyspiraceae and Erysipelotrichales were predominant in responders (n=67), while Enterococcaceae and Lactobacillaceae were, in non-responders (n=137). In BTC pts, Peptostreptococcaceae, Fusobacteriaceae, and Clostridiaceae in LTS; Cardiobacteriaceae, Prevotellaceae, and Actinobacteriota in responders (n=24); and Bacilli in non-responders (n=59) were overrepresented. Conclusions: This interim analysis identified potential biomarkers of the gut microbiota that may contribute to the prognosis and efficacy of chemotherapy in pts with unresectable PC and BTC. The results of this analysis will be validated in the primary analysis.

Purpose: The epithelial-to-mesenchymal transition, the major process by which some cancer cells convert from an epithelial phenotype to a mesenchymal one, has been suggested to drive chemo-resistance and/or metastasis in patients with cancer. However, only a few studies have demonstrated the presence of CD45/CD326 doubly-positive cells (CD45/CD326 DPC) in cancer. We deployed a combination of cell surface markers to elucidate the phenotypic heterogeneity in non–small cell lung cancer (NSCLC) cells and identified a new subpopulation that is doubly-positive for epithelial and non–epithelial cell-surface markers in both NSCLC cells and patients' malignant pleural effusions. Experimental Design: We procured a total of 39 patients' samples, solid fresh lung cancer tissues from 21 patients and malignant pleural effusion samples from 18 others, and used FACS and fluorescence microscopy to check their surface markers. We also examined the EGFR mutations in patients with known acquired EGFR mutations. Results: Our data revealed that 0.4% to 17.9% of the solid tumor tissue cells and a higher percentage of malignant pleural effusion cells harbored CD45/CD326 DPC expressing both epithelial and nonepithelial surface markers. We selected 3 EGFR mutation patients and genetically confirmed that the newly identified cell population really originated from cancer cells. We also found that higher proportions of CD45/CD326 DPC are significantly associated with poor prognosis. Conclusions: In conclusion, varying percentages of CD45/CD326 DPC exist in both solid cancer tissue and malignant pleural effusion in patients with NSCLC. This CD45/CD326 doubly-positive subpopulation can be an important key to clinical management of patients with NSCLC.

The elucidation of dynamic metabolomic changes during gestation is particularly important for the development of methods to evaluate pregnancy status or achieve earlier detection of pregnancy-related complications. Some studies have constructed models to evaluate pregnancy status and predict gestational age using omics data from blood biospecimens; however, less invasive methods are desired. Here we propose a model to predict gestational age, using urinary metabolite information. In our prospective cohort study, we collected 2,741 urine samples from 187 healthy pregnant women, 23 patients with hypertensive disorders of pregnancy, and 14 patients with spontaneous preterm birth. Using gas chromatography-tandem mass spectrometry, we identified 184 urinary metabolites that showed dynamic systematic changes in healthy pregnant women according to gestational age. A model to predict gestational age during normal pregnancy progression was constructed; the correlation coefficient between actual and predicted weeks of gestation was 0.86. The predicted gestational ages of cases with hypertensive disorders of pregnancy exhibited significant progression, compared with actual gestational ages. This is the first study to predict gestational age in normal and complicated pregnancies by using urinary metabolite information. Minimally invasive urinary metabolomics might facilitate changes in the prediction of gestational age in various clinical settings.

The Tohoku Medical Megabank biobank (TMM biobank) is the first major population-based biobank established in Japan. The TMM biobank was established based on two population cohorts and is a reconstruction program from the Great East Japan Earthquake and Tsunami of 2011. The biobank stores more than 3.4 million tubes of biospecimens and associated health and analytic data obtained from approximately 150,000 TMM cohort participants between May 2013 and December 2018, and the TMM biobank currently shares high-quality specimens and data. Various biospecimens, including peripheral and cord blood mononuclear cells, buffy coat, plasma, serum, urine, breast milk and saliva have been collected in the TMM biobank. To minimize human error and maintain the quality of data and specimens, we have been utilizing laboratory information management system into various biobank procedures from registration to storage with various automation systems, such as liquid dispensing, DNA extraction and their storage. The biobank procedures for the quality management system (ISO 9001:2015) and information security management system (ISO 27001:2013) are certified by the International Organization for Standardization. The quality of our biobank samples fulfills the pre-analytical requirements for researchers conducting next-generation whole genome sequencing, DNA array analyses, proteomics, metabolomics, etc. We established analytical centers to conduct standard genomic and multiomic analyses in-house and share the generated data. Additionally, we generate thousands of Epstein-Barr virus (EBV)-transformed lymphoblastoid cell lines and proliferating T cells for functional studies. The TMM biobank serves as an indispensable infrastructure for academic, clinical and industrial research to actualize next-generation medicine in Japan.

Comprehensive genomic profiling enables genomic biomarker detection in advanced solid tumors. Here, to evaluate the utility of circulating tumor DNA (ctDNA) genotyping, we compare trial enrollment using ctDNA sequencing in 1,687 patients with advanced gastrointestinal (GI) cancer in SCRUM-Japan GOZILA (no. UMIN000016343), an observational ctDNA-based screening study, to enrollment using tumor tissue sequencing in the same centers and network (GI-SCREEN, 5,621 patients). ctDNA genotyping significantly shortened the screening duration (11 versus 33 days, P

Clarifying allele frequencies of disease-related genetic variants in a population is important in genomic medicine; however, such data is not yet available for the Japanese population. To estimate frequencies of actionable pathogenic variants in the Japanese population, we examined the reported pathological variants in genes recommended by the American College of Medical Genetics and Genomics (ACMG) in our reference panel of genomic variations, 2KJPN, which was created by whole-genome sequencing of 2049 individuals of the resident cohort of the Tohoku Medical Megabank Project. We searched for pathogenic variants in 2KJPN for 57 autosomal ACMG-recommended genes responsible for 26 diseases and then examined their frequencies. By referring to public databases of pathogenic variations, we identified 143 reported pathogenic variants in 2KJPN for the 57 ACMG recommended genes based on a classification system. At the individual level, 21% of the individuals were found to have at least one reported pathogenic allele. We then conducted a literature survey to review the variants and to check for evidence of pathogenicity. Our results suggest that a substantial number of people have reported pathogenic alleles for the ACMG genes, and reviewing variants is indispensable for constructing the information infrastructure of genomic medicine for the Japanese population.

Although long non-coding RNAs (lncRNAs) are non-protein-coding transcripts by definition, recent studies have shown that a fraction of putative small open reading frames within lncRNAs are translated. However, the biological significance of these hidden polypeptides is still unclear. Here we identify and functionally characterize a novel polypeptide encoded by the lncRNA LINC00961. This polypeptide is conserved between human and mouse, is localized to the late endosome/lysosome and interacts with the lysosomal v-ATPase to negatively regulate mTORC1 activation. This regulation of mTORC1 is specific to activation of mTORC1 by amino acid stimulation, rather than by growth factors. Hence, we termed this polypeptide â small regulatory polypeptide of amino acid response' (SPAR). We show that the SPAR-encoding lncRNA is highly expressed in a subset of tissues and use CRISPR/Cas9 engineering to develop a SPAR-polypeptide-specific knockout mouse while maintaining expression of the host lncRNA. We find that the SPAR-encoding lncRNA is downregulated in skeletal muscle upon acute injury, and using this in vivo model we establish that SPAR downregulation enables efficient activation of mTORC1 and promotes muscle regeneration. Our data provide a mechanism by which mTORC1 activation may be finely regulated in a tissue-specific manner in response to injury, and a paradigm by which lncRNAs encoding small polypeptides can modulate general biological pathways and processes to facilitate tissue-specific requirements, consistent with their restricted and highly regulated expression profile.

Introduction: We performed an exploratory analysis of circulating tumor DNA (ctDNA) in patients (pts) with RAS wild-type (WT) chemotherapy-naïve stage IV metastatic colorectal cancer (mCRC) from the ongoing phase III PARADIGM study (NCT02394834). The clonal and subclonal mutational landscapes of RAS WT pts were analyzed using a mCRC-focused custom ctDNA sequencing panel. Methods: Randomized pts (1:1) received mFOLFOX6 plus either panitumumab or bevacizumab. Pre-treatment plasma samples (DNA yield >10ng/mL and >10nM) were sequenced using the custom ctDNA panel (PlasmaSELECTTM-R 91, PGDx) to detect mCRC mutations, amplifications, and rearrangements in 90, 26, and 3 genes, respectively, as well as microsatellite instability (MSI), in 250kb targeted regions using stringent quality criteria. Pre-treatment archival tissue samples from 590 pts were analyzed by the Broad Institute Solid Tumor panel, which covered 1,072 genes with 7.3Mb targeted regions. Results: Between May 29, 2015 and June 8, 2017, 823 pts were enrolled across 197 sites. Plasma samples were collected from 756 pts, with 747 (98.8%) samples meeting the quality criteria. The average total and distinct sequencing coverages were 34,200 and 5,865, respectively, with 49.1% of bases mapping to regions of interest. Mutation frequencies in ctDNA were generally consistent with those observed in RAS-WT TCGA CRC and rectal cancers (n=283), except for TET2 and DNMT3A potentially due to clonal hematopoiesis. Mutations in BRAF, APC, CTNNB1, PTEN, PIK3CA, and MSI-status observed in ctDNA were significantly different between left and right-sided tumors, consistent with known differences in tumor sidedness. In the 747 plasma samples, 4,072 mutations were observed; 1,871 had a mutant allele frequency (MAF) Conclusion: The validated ultra-deep plasma sequencing panel was concordant with tissue sequencing and detected tumor heterogeneity. The PARADIGM study will report the efficacy of anti-EGFR/VEGF therapies; post-treatment collection of ctDNA is ongoing. The relationship between therapeutic effects and clonal and subclonal mutational landscapes will be examined in the future. Funded by Takeda Pharmaceutical Company Limited, Tokyo, Japan. ClinicalTrial.gov number: NCT02394834 Citation Format: Katsuya Tsuchihara, Riu Yamashita, Takayuki Yoshino, Kohei Shitara, Jun Watanabe, Hirofumi Yasui, Hisatsugu Ohori, Manabu Shiozawa, Kentaro Yamazaki, Eiji Oki, Takeo Sato, Takeshi Naitoh, Yoshito Komatsu, Takeshi Kato, Kazunori Yamanaka, Ikuo Mori, Masamitsu Hihara, Junpei Soeda, Takeharu Yamanaka, Kiwamu Akagi, Atsushi Ochiai, Kei Muro, Victor E. Velculescu, Hiroyuki Uetake. Clonal and subclonal mutational landscapes in circulating tumor DNA in metastatic colorectal cancer: An exploratory analysis from the phase III PARADIGM study [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1974.

The epithelial-to-mesenchymal transition, the major process by which some cancer cells convert from an epithelial phenotype to a mesenchymal one, has been suggested to drive chemo-resistance and/or metastasis in patients with cancer. However, only a few studies have demonstrated the presence of CD45/CD326 doubly-positive cells (CD45/CD326 DPC) in cancer. We deployed a combination of cell surface markers to elucidate the phenotypic heterogeneity in non-small cell lung cancer (NSCLC) cells and identified a new subpopulation that is doubly-positive for epithelial and non-epithelial cell-surface markers in both NSCLC cells and patients' malignant pleural effusions.We procured a total of 39 patients' samples, solid fresh lung cancer tissues from 21 patients and malignant pleural effusion samples from 18 others, and used FACS and fluorescence microscopy to check their surface markers. We also examined theOur data revealed that 0.4% to 17.9% of the solid tumor tissue cells and a higher percentage of malignant pleural effusion cells harbored CD45/CD326 DPC expressing both epithelial and nonepithelial surface markers. We selected 3In conclusion, varying percentages of CD45/CD326 DPC exist in both solid cancer tissue and malignant pleural effusion in patients with NSCLC. This CD45/CD326 doubly-positive subpopulation can be an important key to clinical management of patients with NSCLC.

PurposeA prospective cohort study for pregnant women, the Maternity Log study, was designed to construct a time-course high-resolution reference catalogue of bioinformatic data in pregnancy and explore the associations between genomic and environmental factors and the onset of pregnancy complications, such as hypertensive disorders of pregnancy, gestational diabetes mellitus and preterm labour, using continuous lifestyle monitoring combined with multiomics data on the genome, transcriptome, proteome, metabolome and microbiome.ParticipantsPregnant women were recruited at the timing of first routine antenatal visits at Tohoku University Hospital, Sendai, Japan, between September 2015 and November 2016. Of the eligible women who were invited, 65.4% agreed to participate, and a total of 302 women were enrolled. The inclusion criteria were age ≥20 years and the ability to access the internet using a smartphone in the Japanese language.Findings to dateStudy participants uploaded daily general health information including quality of sleep, condition of bowel movements and the presence of nausea, pain and uterine contractions. Participants also collected physiological data, such as body weight, blood pressure, heart rate and body temperature, using multiple home healthcare devices. The mean upload rate for each lifelog item was ranging from 67.4% (fetal movement) to 85.3% (physical activity), and the total number of data points was over 6 million. Biospecimens, including maternal plasma, serum, urine, saliva, dental plaque and cord blood, were collected for multiomics analysis.Future plansLifelog and multiomics data will be used to construct a time-course high-resolution reference catalogue of pregnancy. The reference catalogue will allow us to discover relationships among multidimensional phenotypes and novel risk markers in pregnancy for the future personalised early prediction of pregnancy complications.

Recent studies have shown that environmental DNA is found almost everywhere. Flower petal surfaces are an attractive tissue to use for investigation of the dispersal of environmental DNA in nature as they are isolated from the external environment until the bud opens and only then can the petal surface accumulate environmental DNA. Here, we performed a crowdsourced experiment, the “Ohanami Project”, to obtain environmental DNA samples from petal surfaces of Cerasus × yedoensis ‘Somei-yoshino’ across the Japanese archipelago during spring 2015. C. × yedoensis is the most popular garden cherry species in Japan and clones of this cultivar bloom simultaneously every spring. Data collection spanned almost every prefecture and totaled 577 DNA samples from 149 collaborators. Preliminary amplicon-sequencing analysis showed the rapid attachment of environmental DNA onto the petal surfaces. Notably, we found DNA of other common plant species in samples obtained across a wide distribution; this DNA likely originated from pollen of the Japanese cedar. Our analysis supports our belief that petal surfaces after blossoming are a promising target to reveal the dynamics of environmental DNA in nature. The success of our experiment also shows that crowdsourced environmental DNA analyses have considerable value in ecological studies.