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Objective: Pancreatic ductal adenocarcinoma (PDAC) stands as one of the most lethal cancers, marked by its lethality and limited treatment options, including the utilisation of checkpoint blockade (ICB) immunotherapy. Epigenetic dysregulation is a defining feature of tumourigenesis that is implicated in immune surveillance, but remains elusive in PDAC., Design: To identify the factors that modulate immune surveillance, we employed in vivo epigenetic-focused CRISPR-Cas9 screen in mouse PDAC tumour models engrafted in either immunocompetent or immunodeficient mice., Results: Here, we identified MED12 as a top hit, emerging as a potent negative modulator of immune tumour microenviroment (TME) in PDAC. Loss of Med12 significantly promoted infiltration and cytotoxicity of immune cells including CD8 + T cells, natural killer (NK) and NK1.1 + T cells in tumours, thereby heightening the sensitivity of ICB treatment in a mouse model of PDAC. Mechanistically, MED12 stabilised heterochromatin protein HP1A to repress H3K9me3-marked endogenous retroelements. The derepression of retrotransposons induced by MED12 loss triggered cytosolic nucleic acid sensing and subsequent activation of type I interferon pathways, ultimately leading to robust inflamed TME . Moreover, we uncovered a negative correlation between MED12 expression and immune resposne pathways, retrotransposon levels as well as the prognosis of patients with PDAC undergoing ICB therapy., Conclusion: In summary, our findings underscore the pivotal role of MED12 in remodelling immnue TME through the epigenetic silencing of retrotransposons, offering a potential therapeutic target for enhancing tumour immunogenicity and overcoming immunotherapy resistance in PDAC., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2024. No commercial re-use. See rights and permissions. Published by BMJ.)

Background: Genomic screening uncovered interferon-gamma (IFNγ) pathway defects in tumours refractory to immune checkpoint blockade (ICB). However, its non-mutational regulation and reversibility for therapeutic development remain less understood., Objective: We aimed to identify ICB resistance-associated druggable histone deacetylases (HDACs) and develop a readily translatable combination approach for patients with hepatocellular carcinoma (HCC)., Design: We correlated the prognostic outcomes of HCC patients from a pembrolizumab trial (NCT03419481) with tumourous cell expressions of all HDAC isoforms by single-cell RNA sequencing. We investigated the therapeutic efficacy and mechanism of action of selective HDAC inhibition in 4 ICB-resistant orthotopic and spontaneous models using immune profiling, single-cell multiomics and chromatin immunoprecipitation-sequencing and verified by genetic modulations and co-culture systems., Results: HCC patients showing higher HDAC1 / 2 / 3 expressions exhibited deficient IFNγ signalling and poorer survival on ICB therapy. Transient treatment of a selective class-I HDAC inhibitor CXD101 resensitised HDAC1/2/3 high tumours to ICB therapies, resulting in CD8 + T cell-dependent antitumour and memory T cell responses. Mechanistically, CXD101 synergised with ICB to stimulate STAT1-driven antitumour immunity through enhanced chromatin accessibility and H3K27 hyperacetylation of IFNγ-responsive genes. Intratumoural recruitment of IFNγ + GZMB + cytotoxic lymphocytes further promoted cleavage of CXD101-induced Gasdermin E (GSDME) to trigger pyroptosis in a STAT1-dependent manner. Notably, deletion of GSDME mimicked STAT1 knockout in abolishing the antitumour efficacy and survival benefit of CXD101-ICB combination therapy by thwarting both pyroptotic and IFNγ responses., Conclusion: Our immunoepigenetic strategy harnesses IFNγ-mediated network to augment the cancer-immunity cycle, revealing a self-reinforcing STAT1-GSDME pyroptotic circuitry as the mechanistic basis for an ongoing phase-II trial to tackle ICB resistance (NCT05873244)., Competing Interests: Competing interests: The authors declare no conflicts of interest related to this work, except for the following declarations. SLC serves as an advisory member for AstraZeneca, MSD, Eisai, BMS, Ipsen and Hengrui, received research funds from MSD, Eisai, Ipsen, SIRTEX and Zailab, and honoraria from AstraZeneca, Eisai, Roche, Ipsen and MSD., (© Author(s) (or their employer(s)) 2024. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Objective: Therapy-induced tumour microenvironment (TME) remodelling poses a major hurdle for cancer cure. As the majority of patients with hepatocellular carcinoma (HCC) exhibits primary or acquired resistance to antiprogrammed cell death (ligand)-1 (anti-PD-[L]1) therapies, we aimed to investigate the mechanisms underlying tumour adaptation to immune-checkpoint targeting., Design: Two immunotherapy-resistant HCC models were generated by serial orthotopic implantation of HCC cells through anti-PD-L1-treated syngeneic, immunocompetent mice and interrogated by single-cell RNA sequencing (scRNA-seq), genomic and immune profiling. Key signalling pathway was investigated by lentiviral-mediated knockdown and pharmacological inhibition, and further verified by scRNA-seq analysis of HCC tumour biopsies from a phase II trial of pembrolizumab (NCT03419481)., Results: Anti-PD-L1-resistant tumours grew >10-fold larger than parental tumours in immunocompetent but not immunocompromised mice without overt genetic changes, which were accompanied by intratumoral accumulation of myeloid-derived suppressor cells (MDSC), cytotoxic to exhausted CD8 + T cell conversion and exclusion. Mechanistically, tumour cell-intrinsic upregulation of peroxisome proliferator-activated receptor-gamma (PPARγ) transcriptionally activated vascular endothelial growth factor-A (VEGF-A) production to drive MDSC expansion and CD8 + T cell dysfunction. A selective PPARγ antagonist triggered an immune suppressive-to-stimulatory TME conversion and resensitised tumours to anti-PD-L1 therapy in orthotopic and spontaneous HCC models. Importantly, 40% (6/15) of patients with HCC resistant to pembrolizumab exhibited tumorous PPARγ induction. Moreover, higher baseline PPARγ expression was associated with poorer survival of anti-PD-(L)1-treated patients in multiple cancer types., Conclusion: We uncover an adaptive transcriptional programme by which tumour cells evade immune-checkpoint targeting via PPARγ/VEGF-A-mediated TME immunosuppression, thus providing a strategy for counteracting immunotherapeutic resistance in HCC., Competing Interests: Competing interests: The authors declare no conflicts of interest that pertain to this work except the following declarations. YMDL is a scientific cofounder of Grail, receives royalties from Illumina, Grail, Sequenom, Xcelom, DRA and Take2, serves as a consultant to Decheng Capital and holds equity in Illumina/Grail, DRA and Take2. SLC serves as a consultant to and receives honoraria from Astra-Zeneca, Eisai and MSD., (© Author(s) (or their employer(s)) 2023. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Objective: Methionine metabolism is involved in a myriad of cellular functions, including methylation reactions and redox maintenance. Nevertheless, it remains unclear whether methionine metabolism, RNA methylation and antitumour immunity are molecularly intertwined., Design: The antitumour immunity effect of methionine-restricted diet (MRD) feeding was assessed in murine models. The mechanisms of methionine and YTH domain-containing family protein 1 (YTHDF1) in tumour immune escape were determined in vitro and in vivo. The synergistic effects of MRD or YTHDF1 depletion with PD-1 blockade were also investigated., Results: We found that dietary methionine restriction reduced tumour growth and enhanced antitumour immunity by increasing the number and cytotoxicity of tumour-infiltrating CD8 + T cells in different mouse models. Mechanistically, the S-adenosylmethionine derived from methionine metabolism promoted the N 6 -methyladenosine (m 6 A) methylation and translation of immune checkpoints, including PD-L1 and V-domain Ig suppressor of T cell activation (VISTA), in tumour cells. Furthermore, MRD or m 6 A-specific binding protein YTHDF1 depletion inhibited tumour growth by restoring the infiltration of CD8 + T cells, and synergised with PD-1 blockade for better tumour control. Clinically, YTHDF1 expression correlated with poor prognosis and immunotherapy outcomes for cancer patients., Conclusions: Methionine and YTHDF1 play a critical role in anticancer immunity through regulating the functions of T cells. Targeting methionine metabolism or YTHDF1 could be a potential new strategy for cancer immunotherapy., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2023. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Background: As the epidemic of COVID-19 is gradually controlled in China, a summary of epidemiological characteristics and interventions may help control its global spread., Methods: Data for COVID-19 cases in Hubei Province (capital, Wuhan) was extracted until 7 March 2020. The spatiotemporal distribution of the epidemic in four periods (before 10 January, 10-22 January, 23 January-6 February and 7 February-7 March) was evaluated, and the impacts of interventions were observed., Results: Among 67 706 COVID-19 cases, 52 111 (76.97%) were aged 30-69 years old, and 34 680 (51.22%) were women. The average daily attack rates (95% CI) were 0.5 (0.3 to 0.7), 14.2 (13.2 to 15.1), 45.7 (44.0 to 47.5) and 8.6 (7.8 to 9.3) cases per 10 6 people in four periods, and the harmonic means (95% CI) of doubling times were 4.28 (4.01 to 4.55), 3.87 (3.78 to 3.98), 5.40 (4.83 to 6.05) and 45.56 (39.70 to 52.80) days. Compared with the first period, daily attack rates rose rapidly in the second period. In the third period, 14 days after 23 January, the daily average attack rate in and outside Wuhan declined by 33.8% and 48.0%; the doubling times increased by 95.0% and 133.2%. In the four periods, 14 days after 7 February, the daily average attack rate in and outside Wuhan decreased by 79.1% and 95.2%; the doubling times increased by 79.2% and 152.0%., Conclusions: The public health interventions were associated with a reduction in COVID-19 cases in Hubei Province, especially in districts outside of Wuhan., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2021. No commercial re-use. See rights and permissions. Published by BMJ.)

Objective: Although a subset of genetic loci have been associated with gastric cancer (GC) risk, the underlying mechanisms are largely unknown. We aimed to identify new susceptibility genes and elucidate their mechanisms in GC development., Design: We conducted a meta-analysis of four genome-wide association studies (GWASs) encompassing 3771 cases and 5426 controls. After targeted sequencing and functional annotation, we performed in vitro and in vivo experiments to confirm the functions of genetic variants and candidate genes. Moreover, we selected 33 promising variants for two-stage replication in 7035 cases and 8323 controls from other five studies., Results: The meta-analysis of GWASs identified three loci at 1q22, 5p13.1 and 10q23.33 associated with GC risk at p<5×10 - 8  and replicated seven known loci at p<0.05. At 5p13.1, the risk rs59133000[C] allele enhanced the binding affinity of NF-κB1 (nuclear factor kappa B subunit 1) to the promoter of PRKAA1 , resulting in a reduced promoter activity and lower expression. The knockout of PRKAA1 promoted both GC cell proliferation and xenograft tumour growth in nude mice. At 10q23.33, the rs3781266[C] and rs3740365[T] risk alleles in complete linkage disequilibrium disrupted and created, respectively, the binding motifs of POU2F1 and PAX3, resulting in an increased enhancer activity and expression of NOC3L , while the NOC3L knockdown suppressed GC cell growth. Moreover, two new loci at 3q11.2 (OR=1.21, p=4.56×10 - 9 ) and 4q28.1 (OR=1.14, p=3.33×10 - 11 ) were associated with GC risk., Conclusion: We identified 12 loci to be associated with GC risk in Chinese populations and deciphered the mechanisms of PRKAA1 at 5p13.1 and NOC3L at 10q23.33 in gastric tumourigenesis., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2020. No commercial re-use. See rights and permissions. Published by BMJ.)

Objective: Hepatocellular carcinoma (HCC), mostly developed in fibrotic/cirrhotic liver, exhibits relatively low responsiveness to immune checkpoint blockade (ICB) therapy. As myeloid-derived suppressor cell (MDSC) is pivotal for immunosuppression, we investigated its role and regulation in the fibrotic microenvironment with an aim of developing mechanism-based combination immunotherapy., Design: Functional significance of MDSCs was evaluated by flow cytometry using two orthotopic HCC models in fibrotic liver setting via carbon tetrachloride or high-fat high-carbohydrate diet and verified by clinical specimens. Mechanistic studies were conducted in human hepatic stellate cell (HSC)-peripheral blood mononuclear cell culture systems and fibrotic-HCC patient-derived MDSCs. The efficacy of single or combined therapy with anti-programmed death-1-ligand-1 (anti-PD-L1) and a clinically trialled BET bromodomain inhibitor i-BET762 was determined., Results: Accumulation of monocytic MDSCs (M-MDSCs), but not polymorphonuclear MDSCs, in fibrotic livers significantly correlated with reduced tumour-infiltrating lymphocytes (TILs) and increased tumorigenicity in both mouse models. In human HCCs, the tumour-surrounding fibrotic livers were markedly enriched with M-MDSC, with its surrogate marker CD33 significantly associated with aggressive tumour phenotypes and poor survival rates. Mechanistically, activated HSCs induced monocyte-intrinsic p38 MAPK signalling to trigger enhancer reprogramming for M-MDSC development and immunosuppression. Treatment with p38 MAPK inhibitor abrogated HSC-M-MDSC crosstalk to prevent HCC growth. Concomitant with patient-derived M-MDSC suppression by i-BET762, combined treatment with anti-PD-L1 synergistically enhanced TILs, resulting in tumour eradication and prolonged survival in the fibrotic-HCC mouse model., Conclusion: Our results signify how non-tumour-intrinsic properties in the desmoplastic microenvironment can be exploited to reinstate immunosurveillance, providing readily translatable combination strategies to empower HCC immunotherapy., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2020. No commercial re-use. See rights and permissions. Published by BMJ.)

Objective: To monitor trastuzumab resistance and determine the underlying mechanisms for the limited response rate and rapid emergence of resistance of HER2+ metastatic gastric cancer (mGC)., Design: Targeted sequencing of 416 clinically relevant genes was performed in 78 paired plasma and tissue biopsy samples to determine plasma-tissue concordance. Then, we performed longitudinal analyses of 97 serial plasma samples collected from 24 patients who were HER2+  to track the resistance during trastuzumab treatment and validated the identified candidate resistance genes., Results: The results from targeted sequencing-based detection of somatic copy number alterations (SCNA) of HER2 gene were highly consistent with fluorescence in situ hybridisation data, and the detected HER2 SCNA was better than plasma carcinoembryonic antigen levels at predicting tumour shrinkage and progression. Furthermore, most patients with innate trastuzumab resistance presented high HER2 SCNA during progression compared with baseline, while HER2 SCNA decreased in patients with acquired resistance. PIK3CA mutations were significantly enriched in patients with innate resistance, and ERBB2/4 genes were the most mutated genes, accounting for trastuzumab resistance in six (35.3%) and five (29.4%) patients in baseline and progression plasma, respectively. Patients with PIK3CA/R1/C3 or ERBB2/4 mutations in the baseline plasma had significantly worse progression-free survival. Additionally, mutations in NF1 contributed to trastuzumab resistance, which was further confirmed through in vitro and in vivo studies, while combined HER2 and MEK/ERK blockade overcame trastuzumab resistance., Conclusion: Longitudinal circulating tumour DNA sequencing provides novel insights into gene alterations underlying trastuzumab resistance in HER2+mGC., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2019. No commercial re-use. See rights and permissions. Published by BMJ.)

Objective: Although evidence suggests an inverse association between calcium intake and the risk of colorectal cancer, the mechanisms remain unclear. The calcium-sensing receptor (CASR) is expressed abundantly in normal colonic epithelium and may influence carcinogenesis. We hypothesized that calcium intake might be associated with lower risk of CASR-positive, but not CASR-negative, colorectal cancer., Design: We assessed tumour CASR protein expression using immunohistochemistry in 779 incident colon and rectal cancer cases that developed among 136 249 individuals in the Nurses' Health Study and Health Professionals Follow-Up Study. Duplication method Cox proportional hazards regression analysis was used to assess associations of calcium intake with incidence of colorectal adenocarcinoma subtypes by CASR status., Results: Total calcium intake was inversely associated with the risk of developing colorectal cancer (p trend =0.01, comparing ≥1200 vs <600 mg/day: multivariable HR=0.75, 95% CI 0.60 to 0.95). For the same comparison, higher total calcium intake was associated with a lower risk of CASR-positive tumours (p trend =0.003, multivariable HR=0.67, 95% CI 0.51 to 0.86) but not with CASR-negative tumours (p trend =0.67, multivariable HR=1.15, 95% CI 0.75 to 1.78; p heterogeneity =0.06 between the CASR subtypes). The stronger inverse associations of calcium intake with CASR-positive but not CASR-negative tumours generally appeared consistent regardless of sex, tumour location and source of calcium., Conclusions: Our molecular pathological epidemiology data suggest a causal relationship between higher calcium intake and lower colorectal cancer risk, and a potential role of CASR in mediating antineoplastic effect of calcium., Competing Interests: Competing interests: None declared., (© Article author(s) (or their employer(s) unless otherwise stated in the text of the article) 2018. All rights reserved. No commercial use is permitted unless otherwise expressly granted.)

Background: Neutrophils are accumulated in inflamed mucosa of IBD and play an important role in the pathogenesis. CD177 is expressed in neutrophils specifically and upregulated during inflammation. However, the role of CD177 + neutrophils in pathogenesis of IBD remains elusive., Materials and Methods: Expression of CD177 was analysed in peripheral blood and intestinal mucosa from patients with IBD using quantitative RT-PCR, flow cytometry and immunohistochemistry. CD177 + and CD177 - neutrophils were isolated to determine gene differences by RNA sequencing. Colitis was established in CD177 -/- and wild-type mice in response to dextran sulfate sodium (DSS) insults to determine the role of CD177 + neutrophils in IBD., Results: CD177 + neutrophils were markedly increased in peripheral blood and inflamed mucosa from patients with active IBD compared with healthy controls. RNA sequencing revealed that differential gene expression between CD177 + and CD177 - neutrophils from patients with IBD was associated with response to bacterial defence, hydrogen peroxide and reactive oxygen species (ROS). CD177 + neutrophils produced lower levels of proinflammatory cytokines (ie, interferon-γ, interleukin (IL)-6, IL-17A), but higher levels of IL-22 and transforming growth factor-β, and exhibited increased bactericidal activities (ie, ROS, antimicrobial peptides, neutrophil extracellular trap) compared with CD177 - subset. CD177 -/- mice developed more severe colitis on DSS insults compared with wild-type mice. Moreover, CD177 deficiency led to compromised intestinal barrier and impaired antibacterial immunity through decreased production of IL-22 by CD177 - neutrophils., Conclusions: CD177 + neutrophils represent functionally activated population and play a protective role in IBD through increased bactericidal activity and IL-22 production. Targeting CD177 + neutrophils may be beneficial for treatment of IBD., Competing Interests: Competing interests: None declared., (Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://www.bmj.com/company/products-services/rights-and-licensing/.)

Objective: Liver tumour-initiating cells (T-ICs) are critical for hepatocarcinogenesis. However, the underlying mechanism regulating the function of liver T-ICs remains unclear., Methods: Tissue microarrays containing 242 hepatocellular carcinoma (HCC) samples were used for prognostic analysis. Magnetically activated cell sorting was used to isolate epithelial cell adhesion molecule (EPCAM)-positive cells. The gene expressions affected by miR-429 were determined by arrays. Co-immunoprecipitation was used to study interactions among retinoblastoma protein (RB1), Rb binding protein 4 (RBBP4) and E2F transcription factor 1 (E2F1). The DNA methylation status in CpG islands was detected by quantitative methylation analysis. miRNAs in microvesicles were isolated by a syringe filter system., Results: The significant prognosis factor miR-429 was upregulated in HCC tissues and also in primary liver T-ICs isolated from clinical samples. The enrichment of miR-429 in EPCAM+ T-ICs contributed to hepatocyte self-renewal, malignant proliferation, chemoresistance and tumorigenicity. A novel functional axis involving miR-429, RBBP4, E2F1 and POU class 5 homeobox 1 (POU5F1 or OCT4) governing the regulation of liver EPCAM+ T-ICs was established in vitro and in vivo. The molecular mechanism regulating miR-429 expression, involving four abnormal hypomethylated sites upstream of the miR-200b/miR-200a/miR-429 cluster, was first defined in both EPCAM+ liver T-ICs and very early-stage HCC tissues. miR-429 secreted by high-expressing cells has the potential to become a proactive signalling molecule to mediate intercellular communication., Conclusions: Epigenetic modification of miR-429 can manipulate liver T-ICs by targeting the RBBP4/E2F1/OCT4 axis. This miRNA might be targeted to inactivate T-ICs, thus providing a novel strategy for HCC prevention and treatment., (Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://group.bmj.com/group/rights-licensing/permissions.)

Background: An increasing USA population, especially as a result of foreign immigration, has been the recent focus of healthcare research and policy debate. However, little is known about domestic immigration and its impact on the measures of health conditions and behaviours used to develop and evaluate public health policies and programmes. The objective of this research is to compare health conditions and behaviours between domestic immigrants and established residents in Nevada, the fastest-growing state in the USA., Methods: The Nevada 2004-2006 Behavioural Risk Factor Surveillance Survey and added state residency questions were used to examine the associations between length of residence and 24 explanatory variables on health behaviours and conditions. Weighted multiple logistic regressions were used for data analyses., Results: Among the 8663 respondents weighted to the total Nevada population, long-term residents (5 to <10 years), compared with newcomers or short-term residents, were more likely to report fair or poor health (OR=1.98, 95% CI 1.15 to 3.40) and obesity (OR=2.18, 95% CI 1.10 to 4.32), and to live with firearms in the home (OR=6.34, 95% CI 1.75 to 22.9). They were less likely to report having diabetes (OR=0.49, 95% CI 0.25 to 0.96), having had an HIV test (OR=0.60 95% CI 0.41 to 0.89), having prostate cancer (OR=0.20 95% CI 0.04 to 0.93) or having had a mammogram (OR=0.41, 95% CI 0.25 to 0.70)., Conclusions: Long-term residents, compared with newcomers, provide a unique comparison group that share the same physical and policy environment but in different time periods. Knowledge of differences such as these can improve the specificity in identifying target groups for particular interventions.