Project description:Pulmonary enteric adenocarcinoma (PEAD) is a rare non-small cell lung cancer subtype. It is poorly characterized and cannot be distinguished from metastatic colorectal or upper gastrointestinal adenocarcinomas (ADCs) by means of routine pathological methods. As DNA methylation patterns are known to be highly tissue specific, we aimed to develop a methylation-based algorithm to differentiate these entities. To this end, genome wide methylation profiles of 600 primary pulmonary, colorectal and upper gastrointestinal ADCs obtained from The Cancer Genome Atlas (TCGA) and the Gene Expression Omnibus (GEO) database were used as a reference cohort to train a machine learning algorithm. The resulting classifier correctly classified all samples from a validation cohort consisting of 680 primary pulmonary, colorectal and upper gastrointestinal ADCs from TCGA and the GEO database, demonstrating the ability of the algorithm to reliably distinguish these three entities. We then analyzed DNA methylation data of 15 PEADs as well as four pulmonary metastases and four primary colorectal ADCs with the algorithm. All 15 PEADs were reliably classified as primary pulmonary tumors and all four metastases as well as all four primary colorectal ADC samples were identified as primary colorectal ADCs. In a t-distributed stochastic neighbor embedding analysis, the PEAD samples did not form a separate methylation subclass but rather diffusely intermixed with other pulmonary ADCs. Additional characterization of the PEAD series using fluorescence in-situ hybridization, next generation sequencing and copy number analysis revealed KRAS mutations in nine of 15 samples (60%) and a high number of structural chromosomal changes. Except for an unusually high rate of chromosome 20 gain (66.7%) the molecular data was mostly reminiscent of standard pulmonary ADCs. In conclusion, we provide sound evidence of the pulmonary origin of PEAD and in addition provide a publicly available machine learning based algorithm to reliably distinguish PEAD from metastatic colorectal cancer and upper gastrointestinal adenocarcinomas.

Project description:BackgroundAs a rare subtype of lung adenocarcinoma, the diagnosis of pulmonary enteric adenocarcinoma (PEAC) remains challenging due to overlapping morphologic spectrum with lung metastatic colorectal cancer (lmCRC). However, the molecular features of PEAC as a separate lung cancer entity are poorly understood.MethodsWe performed whole-exome sequencing and targeted bisulfite sequencing of 32 PEAC and 30 lmCRC to improve differential molecular characterization of the two diseases. We used machine learning methods to select key markers and developed a diagnostic classifier. In addition, we validated the classifier in the internal test cohort and an independently recruited external validation cohort with 17 PEAC and 7 lmCRC.FindingsOur results showed that EGFR was the key driver mutation in PEAC but at a lower prevalence compared to typical lung adenocarcinomas, whereas ERBB2 and KRAS were more frequently observed in PEAC. By contrast, we observed significant enrichment of KRAS and APC mutations in lmCRC compared with PEAC. At the chromosome arm level, copy number variations in 13q, 14q, and 18p were the major chromosomal differences observed between PEAC and lmCRC. Furthermore, by comparing differentially methylated regions (DMRs), we established a neat DNA methylation-based classifier consisting of eight DMRs. This classifier correctly classified all samples in the training cohort and 95% of the samples in the internal test cohort. An external validation cohort of 24 cases recruited from multiple centers in China also reliably agreed with pathological diagnosis.InterpretationThese results provide solid evidence of PEAC-specific genomic characteristics and demonstrate the potential utility of DNA methylation markers for auxiliary diagnosis of PEAC and lmCRC.FundingThis work was supported by National key research and development project 2019YFC1315700, CAMS Key Laboratory of Translational Research on Lung Cancer (2018PT31035), and Beijing Natural Science Foundation (7222144).

Project description:BACKGROUND Enteric glia contribute to the pathophysiology of various intestinal immune-driven diseases, such as postoperative ileus (POI), a motility disorder and common complication after abdominal surgery. Enteric gliosis of the intestinal muscularis externa (ME) has been identified as part of POI development. However, the glia-restricted responses and activation mechanisms are poorly understood. The sympathetic nervous system becomes rapidly activated by abdominal surgery. It modulates intestinal immunity, innervates all intestinal layers, and directly interfaces with enteric glia. We hypothesized that sympathetic innervation controls enteric glia reactivity in response to surgical trauma. METHODS Sox10iCreERT2/Rpl22HA/+ mice were subjected to a mouse model of laparotomy or intestinal manipulation to induce POI. Histological, protein, and transcriptomic analyses were performed to analyze glia-specific responses. Interactions between the sympathetic nervous system and enteric glia were studied in mice chemically depleted of TH+ sympathetic neurons and glial-restricted Sox10iCreERT2/JellyOPfl/+/Rpl22HA/+ mice, allowing optogenetic stimulation of β-adrenergic downstream signaling and glial-specific transcriptome analyses. A laparotomy model was used to study the effect of sympathetic signaling on enteric glia in the absence of intestinal manipulation. Mechanistic studies included adrenergic receptor expression profiling in vivo and in vitro and adrenergic agonism treatments of primary enteric glial cell cultures to elucidate the role of sympathetic signaling in acute enteric gliosis and POI. RESULTS With ~4000 differentially expressed genes, the most substantial enteric glia response occurs early after intestinal manipulation. During POI, enteric glia switch into a reactive state and continuously shape their microenvironment by releasing inflammatory and migratory factors. Sympathetic denervation reduced the inflammatory response of enteric glia in the early postoperative phase. Optogenetic and pharmacological stimulation of β-adrenergic downstream signaling triggered enteric glia reactivity. Finally, distinct adrenergic agonists revealed β-1/2 adrenoceptors as the molecular targets of sympathetic–driven enteric glial reactivity. CONCLUSIONS Enteric glia act as early responders during post-traumatic intestinal injury and inflammation. Intact sympathetic innervation and active β-adrenergic receptor signaling in enteric glia is a trigger of the immediate glial postoperative inflammatory response. With immune-activating cues originating from the sympathetic nervous system as early as the initial surgical incision, adrenergic signaling in enteric glia presents a promising target for preventing POI development.

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DNA methylation signature reliably distinguishes pulmonary enteric adenocarcinoma from metastatic colorectal cancer

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