Hepatic gap junctions amplify alcohol liver injury by propagating cGAS-mediated IRF3 activation.

Alcohol-related liver disease (ALD) accounts for the majority of cirrhosis and liver-related deaths worldwide. Activation of IFN-regulatory factor (IRF3) initiates alcohol-induced hepatocyte apoptosis, which fuels a robust secondary inflammatory response that drives ALD. The dominant molecular mechanism by which alcohol activates IRF3 and the pathways that amplify inflammatory signals in ALD remains unknown. Here we show that cytoplasmic sensor cyclic guanosine monophosphate-adenosine monophosphate (AMP) synthase (cGAS) drives IRF3 activation in both alcohol-injured hepatocytes and the neighboring parenchyma via a gap junction intercellular communication pathway. Hepatic RNA-seq analysis of patients with a wide spectrum of ALD revealed that expression of the cGAS-IRF3 pathway correlated positively with disease severity. Alcohol-fed mice demonstrated increased hepatic expression of the cGAS-IRF3 pathway. Mice genetically deficient in cGAS and IRF3 were protected against ALD. Ablation of cGAS in hepatocytes only phenocopied this hepatoprotection, highlighting the critical role of hepatocytes in fueling the cGAS-IRF3 response to alcohol. We identified connexin 32 (Cx32), the predominant hepatic gap junction, as a critical regulator of spreading cGAS-driven IRF3 activation through the liver parenchyma. Disruption of Cx32 in ALD impaired IRF3-stimulated gene expression, resulting in decreased hepatic injury despite an increase in hepatic steatosis. Taken together, these results identify cGAS and Cx32 as key factors in ALD pathogenesis and as potential therapeutic targets for hepatoprotection.

Germline mutations in oncogene-induced senescence pathways are associated with multiple sessile serrated adenomas.

BACKGROUND & AIMS: Little is known about the genetic factors that contribute to the development of sessile serrated adenomas (SSAs). SSAs contain somatic mutations in BRAF or KRAS early in development. However, evidence from humans and mouse models indicates that these mutations result in oncogene-induced senescence (OIS) of intestinal crypt cells. Progression to serrated neoplasia requires cells to escape OIS via inactivation of tumor suppressor pathways. We investigated whether subjects with multiple SSAs carry germline loss-of function mutations (nonsense and splice site) in genes that regulate OIS: the p16-Rb and ATM-ATR DNA damage response pathways. METHODS: Through a bioinformatic analysis of the literature, we identified a set of genes that function at the main nodes of the p16-Rb and ATM-ATR DNA damage response pathways. We performed whole-exome sequencing of 20 unrelated subjects with multiple SSAs; most had features of serrated polyposis. We compared sequences with those from 4300 subjects matched for ethnicity (controls). We also used an integrative genomics approach to identify additional genes involved in senescence mechanisms. RESULTS: We identified mutations in genes that regulate senescence (ATM, PIF1, TELO2,XAF1, and RBL1) in 5 of 20 subjects with multiple SSAs (odds ratio, 3.0; 95% confidence interval, 0.9­8.9; P =.04). In 2 subjects,we found nonsense mutations in RNF43, indicating that it is also associated with multiple serrated polyps (odds ratio, 460; 95% confidence interval, 23.1­16,384; P = 6.8 x 10(-5)). In knockdown experiments with pancreatic duct cells exposed to UV light, RNF43 appeared to function as a regulator of ATMATRDNA damage response. CONCLUSIONS: We associated germline loss-of-function variants in genes that regulate senescence pathways with the development of multiple SSAs.We identified RNF43 as a regulator of the DNA damage response and associated nonsense variants in this gene with a high risk of developing SSAs.

Recurrences are common after endoscopic ampullectomy for adenoma in the familial adenomatous polyposis (FAP) syndrome.

BACKGROUND: Endoscopic ampullectomy is increasingly performed in patients with familial adenomatous polyposis (FAP)-associated ampullary adenomas. We sought to define the procedure-associated morbidities and long-term outcomes. METHODS: We performed a retrospective chart review of patients with FAP who underwent endoscopic ampullectomy at two tertiary institutions between 1999 and 2010. The severity of duodenal polyposis was classified according to Spigelman's classification. RESULTS: Of 26 FAP patients who underwent endoscopic ampullectomy, 21 arose in the setting of Spigelman's stage II duodenal polyposis. Adverse events associated with endoscopic ampullectomy included acute pancreatitis (19.2%), abdominal pain (7.6%), and bleeding (3.8%). The mean resected adenoma size was 0.99 ± 0.34 cm. Three adenomas (12.0%) contained foci of high-grade dysplasia. Follow-up data were available for 24 patients. The mean follow-up duration was 84.5 ± 36.2 months. Adenoma recurrence was observed in 14 patients (58.3%; 14/24) at a mean of 38.3 months after initial ampullectomy. Adenomas ≥10 mm recurred more frequently than smaller adenomas (76.9 vs. 36.4%; p = 0.002). Positive margins were not associated with higher recurrence rates. No cancers were observed during long-term follow-up. Three patients underwent a Whipple procedure, but none was performed for a recurrent ampullary adenoma. CONCLUSIONS: Endoscopic ampullectomy in FAP can be performed safely. Because ampullary adenomas frequently recur after endoscopic ampullectomy, close surveillance is essential. Smaller tumors are less likely to recur, suggesting a benefit for early recognition of these lesions.

Development of a clinical polygenic risk score assay and reporting workflow.

Implementation of polygenic risk scores (PRS) may improve disease prevention and management but poses several challenges: the construction of clinically valid assays, interpretation for individual patients, and the development of clinical workflows and resources to support their use in patient care. For the ongoing Veterans Affairs Genomic Medicine at Veterans Affairs (GenoVA) Study we developed a clinical genotype array-based assay for six published PRS. We used data from 36,423 Mass General Brigham Biobank participants and adjustment for population structure to replicate known PRS-disease associations and published PRS thresholds for a disease odds ratio (OR) of 2 (ranging from 1.75 (95% CI: 1.57-1.95) for type 2 diabetes to 2.38 (95% CI: 2.07-2.73) for breast cancer). After confirming the high performance and robustness of the pipeline for use as a clinical assay for individual patients, we analyzed the first 227 prospective samples from the GenoVA Study and found that the frequency of PRS corresponding to published OR > 2 ranged from 13/227 (5.7%) for colorectal cancer to 23/150 (15.3%) for prostate cancer. In addition to the PRS laboratory report, we developed physician- and patient-oriented informational materials to support decision-making about PRS results. Our work illustrates the generalizable development of a clinical PRS assay for multiple conditions and the technical, reporting and clinical workflow challenges for implementing PRS information in the clinic.

Case Report: A Rare Case of Esophagogastric Junctional Squamous Cell Carcinoma After the Successful Treatment of Neuroendocrine Carcinoma: Clonal Tumor Evolution Revealed by Genetic Analysis.

Neuroendocrine carcinoma (NEC) of the esophagogastric junction (EGJ) is a rare disease with no established treatments. Herein, we describe a case of recurrent squamous cell carcinoma (SCC) after achieving complete response to chemotherapy against NEC of the EGJ. A 67-year-old man was referred to our hospital because of epigastric discomfort. Computed tomography imaging and esophagogastroduodenoscopy revealed ulcerated tumors at the EGJ. Endoscopic biopsy revealed small tumor cells with a high nuclear/cytoplasmic ratio, suggesting small-cell NEC. Immunohistochemistry (IHC) analysis showed tumor cells with an MIB-1 index of 80%. The patient achieved complete response after 10 cycles of chemotherapy. Follow-up endoscopic examination revealed small red-colored mucosal lesions in the center of the cicatrized primary lesion. Re-biopsy detected cancer cells harboring large eosinophilic cytoplasm with keratinization and no evidence of NEC components. IHC of the cells were cytokeratin 5/6-positive and p53-negative. The tumor persisted without evidence of metastases after chemoradiotherapy, and total gastrectomy with lymph node dissection was performed. Pathological assessment of the resected specimens revealed SCC, without evidence of NEC. The patient survived without a recurrence for >3 years after the initial presentation. Somatic mutation profiles of the primary NEC and recurrent SCC were analyzed by targeted amplicon sequencing covering common cancer-related mutations. Both tumors possessed TP53 Q192X mutation, whereas SMAD4 S517T was found only in SCC, suggesting that both tumor components originated from a founder clone with a stop-gain mutation in TP53. The somatic mutation profile of the tumors indicated that that loss of heterozygosity (LOH) at the TP53 gene might have occurred during the differentiation of the founder clone into NEC, while a SMAD4 mutation might have contributed to SCC development, indicating branching and subclonal evolution from common founder clone to both NEC and SCC. The mutation assessments provided valuable information to better understand the clonal evolution of metachronous cancers.

Homologous Recombination Repair Truncations Predict Hypermutation in Microsatellite Stable Colorectal and Endometrial Tumors.

INTRODUCTION: Somatic mutations in BRCA1/2 and other homologous recombination repair (HRR) genes have been associated with sensitivity to PARP inhibitors and/or platinum agents in several cancers, whereas hypermutant tumors caused by alterations in POLE or mismatch repair genes have demonstrated robust responses to immunotherapy. We investigated the relationship between somatic truncations in HRR genes and hypermutation in colorectal cancer (CRC) and endometrial cancer (EC). METHODS: We analyzed the mutational spectra associated with somatic BRCA1/2 truncations in multiple genomic cohorts (N = 2,335). From these results, we devised a classifier incorporating HRR genes to predict hypermutator status among microsatellite stable (MSS) tumors. Using additional genomic cohorts (N = 1,439) and functional in vivo assays, we tested the classifier to disambiguate POLE variants of unknown significance and identify MSS hypermutators without somatic POLE exonuclease domain mutations. RESULTS: Hypermutator phenotypes were prevalent among CRCs with somatic BRCA1/2 truncations (50/62, 80.6%) and ECs with such mutations (44/47, 93.6%). The classifier predicted MSS hypermutators with a cumulative true-positive rate of 100% in CRC and 98.0% in EC and a false-positive rate of 0.07% and 0.63%. Validated by signature analyses of tumor exomes and in vivo assays, the classifier accurately reassigned multiple POLE variants of unknown significance as pathogenic and identified MSS hypermutant samples without POLE exonuclease domain mutations. DISCUSSION: Somatic truncations in HRR can accurately fingerprint MSS hypermutators with or without known pathogenic exonuclease domain mutations in POLE and may serve as a low-cost biomarker for immunotherapy decisions in MSS CRC and EC.

Colorectal cancer susceptibility variants and risk of conventional adenomas and serrated polyps: results from three cohort studies.

BACKGROUND: Increasing evidence suggests that conventional adenomas (CAs) and serrated polyps (SPs) represent two distinct groups of precursor lesions for colorectal cancer (CRC). The influence of common genetic variants on risk of CAs and SPs remain largely unknown. METHODS: Among 27 426 participants within three prospective cohort studies, we created a weighted genetic risk score (GRS) based on 40 CRC-related single nucleotide polymorphisms (SNPs) identified in previous genome-wide association studies; and we examined the association of GRS (per one standard deviation increment) with risk of CAs, SPs and synchronous CAs and SPs, by multivariable logistic regression. We also analysed individual variants in the secondary analysis. RESULTS: During 18-20 years of follow-up, we documented 2952 CAs, 1585 SPs and 794 synchronous CAs and SPs. Higher GRS was associated with increased risk of CAs [odds ratio (OR) = 1.17, 95% confidence interval (CI): 1.12-1.21] and SPs (OR = 1.09, 95% CI: 1.03-1.14), with a stronger association for CAs than SPs (Pheterogeneity=0.01). An even stronger association was found for patients with synchronous CAs and SPs (OR = 1.32), advanced CAs (OR = 1.22) and multiple CAs (OR = 1.25). Different sets of variants were associated with CAs and SPs, with a Spearman correlation coefficient of 0.02 between the ORs associating the 40 SNPs with the two lesions. After correcting for multiple testing, three variants were associated with CAs (rs3802842, rs6983267 and rs7136702) and two with SPs (rs16892766 and rs4779584). CONCLUSIONS: Common genetic variants play a potential role in the conventional and serrated pathways of CRC. Different sets of variants are identified for the two pathways, further supporting the aetiological heterogeneity of CRC.

Effect of Low-dose and Standard-dose Aspirin on PGE2 Biosynthesis Among Individuals with Colorectal Adenomas: A Randomized Clinical Trial.

Low-dose aspirin is recommended by the U.S. Preventive Services Task Force for primary prevention of colorectal cancer in certain individuals. However, broader implementation will require improved precision prevention approaches to identify those most likely to benefit. The major urinary metabolite of PGE2, 11α-hydroxy-9,15-dioxo-2,3,4,5-tetranor-prostane-1,20-dioic acid (PGE-M), is a biomarker for colorectal cancer risk, but it is unknown whether PGE-M is modifiable by aspirin in individuals at risk for colorectal cancer. Adults (N = 180) who recently underwent adenoma resection and did not regularly use aspirin or NSAIDs were recruited to a double-blind, placebo-controlled, randomized trial of aspirin at 81 or 325 mg/day for 8-12 weeks. The primary outcome was postintervention change in urinary PGE-M as measured by LC/MS. A total of 169 participants provided paired urine samples for analysis. Baseline PGE-M excretion was 15.9 ± 14.6 (mean ± S.D, ng/mg creatinine). Aspirin significantly reduced PGE-M excretion (-4.7 ± 14.8) compared with no decrease (0.8 ± 11.8) in the placebo group (P = 0.015; mean duration of treatment = 68.9 days). Aspirin significantly reduced PGE-M levels in participants receiving either 81 (-15%; P = 0.018) or 325 mg/day (-28%; P < 0.0001) compared with placebo. In 40% and 50% of the individuals randomized to 81 or 325 mg/day aspirin, respectively, PGE-M reduction reached a threshold expected to prevent recurrence in 10% of individuals. These results support that aspirin significantly reduces elevated levels of PGE-M in those at increased colorectal cancer risk to levels consistent with lower risk for recurrent neoplasia and underscore the potential utility of PGE-M as a precision chemoprevention biomarker. The ASPIRED trial is registered as NCT02394769.

Hepatic connexin 32 associates with nonalcoholic fatty liver disease severity.

Emerging data highlight the critical role for the innate immune system in the progression of nonalcoholic fatty liver disease (NAFLD). Connexin 32 (Cx32), the primary liver gap junction protein, is capable of modulating hepatic innate immune responses and has been studied in dietary animal models of steatohepatitis. In this work, we sought to determine the association of hepatic Cx32 with the stages of human NAFLD in a histologically characterized cohort of 362 patients with NAFLD. We also studied the hepatic expression of the genes and proteins known to interact with Cx32 (known as the connexome) in patients with NAFLD. Last, we used three independent dietary mouse models of nonalcoholic steatohepatitis to investigate the role of Cx32 in the development of steatohepatitis and fibrosis. In a univariate analysis, we found that Cx32 hepatic expression associates with each component of the NAFLD activity score and fibrosis severity. Multivariate analysis revealed that Cx32 expression most closely associated with the NAFLD activity score and fibrosis compared to known risk factors for the disease. Furthermore, by analyzing the connexome, we identified novel genes related to Cx32 that associate with NAFLD progression. Finally, we demonstrated that Cx32 deficiency protects against liver injury, inflammation, and fibrosis in three murine models of nonalcoholic steatohepatitis by limiting initial diet-induced hepatoxicity and subsequent increases in intestinal permeability. Conclusion: Hepatic expression of Cx32 strongly associates with steatohepatitis and fibrosis in patients with NAFLD. We also identify novel genes associated with NAFLD and suggest that Cx32 plays a role in promoting NAFLD development. (Hepatology Communications 2018;2:786-797).

Continuity of transcriptomes among colorectal cancer subtypes based on meta-analysis.

BACKGROUND: Previous approaches to defining subtypes of colorectal carcinoma (CRC) and other cancers based on transcriptomes have assumed the existence of discrete subtypes. We analyze gene expression patterns of colorectal tumors from a large number of patients to test this assumption and propose an approach to identify potentially a continuum of subtypes that are present across independent studies and cohorts. RESULTS: We examine the assumption of discrete CRC subtypes by integrating 18 published gene expression datasets and > 3700 patients, and contrary to previous reports, find no evidence to support the existence of discrete transcriptional subtypes. Using a meta-analysis approach to identify co-expression patterns present in multiple datasets, we identify and define robust, continuously varying subtype scores to represent CRC transcriptomes. The subtype scores are consistent with established subtypes (including microsatellite instability and previously proposed discrete transcriptome subtypes), but better represent overall transcriptional activity than do discrete subtypes. The scores are also better predictors of tumor location, stage, grade, and times of disease-free survival than discrete subtypes. Gene set enrichment analysis reveals that the subtype scores characterize T-cell function, inflammation response, and cyclin-dependent kinase regulation of DNA replication. CONCLUSIONS: We find no evidence to support discrete subtypes of the CRC transcriptome and instead propose two validated scores to better characterize a continuity of CRC transcriptomes.

Health Care Infrastructure for Financially Sustainable Clinical Genomics.

Next-generation sequencing has evolved technically and economically into the method of choice for interrogating the genome in cancer and inherited disorders. The introduction of procedural code sets for whole-exome and genome sequencing is a milestone toward financially sustainable clinical implementation; however, achieving reimbursement is currently a major challenge. As part of a prospective quality-improvement initiative to implement the new code sets, we adopted Agile, a development methodology originally devised in software development. We implemented eight functionally distinct modules (request review, cost estimation, preauthorization, accessioning, prebilling, testing, reporting, and reimbursement consultation) and obtained feedback via an anonymous survey. We managed 50 clinical requests (January to June 2015). The fraction of pursued-to-requested cases (n = 15/50; utilization management fraction, 0.3) aimed for a high rate of preauthorizations. In 13 of 15 patients the insurance plan required preauthorization, which we obtained in 70% and ultimately achieved reimbursement in 50%. Interoperability enabled assessment of 12 different combinations of modules that underline the importance of an adaptive workflow and policy tailoring to achieve higher yields of reimbursement. The survey confirmed a positive attitude toward self-organizing teams. We acknowledge the individuals and their interactions and termed the infrastructure: human pipeline. Nontechnical barriers currently are limiting the scope and availability of clinical genomic sequencing. The presented human pipeline is one approach toward long-term financial sustainability of clinical genomics.

Molecular pathways: aspirin and Wnt signaling-a molecularly targeted approach to cancer prevention and treatment.

The anti-inflammatory properties of aspirin have resulted in its widespread use as an analgesic, antipyretic, and cardioprotective agent. Beyond these applications, multiple observational studies and randomized controlled trials have demonstrated a chemopreventative role for aspirin, particularly in the development of colorectal neoplasia. Given the critical importance of Wnt dysregulation in colorectal carcinogenesis, the interplay between aspirin and canonical Wnt signaling has become a focus of investigation. These studies have illuminated our understanding of the anticancer mechanisms of aspirin, yielding the identification of potential biomarkers for which aspirin's chemopreventative efficacy can be safely optimized into routine clinical practice and providing leads into the discovery of novel preventive and therapeutic targets. In this review, we summarize key experimental and clinical studies of this interaction, as well as highlighting future strategies to advance their clinical translation.

TFF2-CXCR4 Axis Is Associated with BRAF V600E Colon Cancer.

Oncogene-induced senescence (OIS), a tumor-suppressive mechanism that is induced by the replicative and metabolic stress of oncogene activation, is a key barrier in the development of BRAF V600E colon cancer. Inhibition of this mechanism has been observed through epigenetic changes observed in sporadic serrated polyps, as well as through the germline mutations associated with those who develop serrated polyposis. We hypothesize that upregulated autocrine factors exist that are specific to the serrated pathway and also promote bypass of oncogene-induced senescence. To identify such autocrine factors, we integrate analyses of microarrays of sessile serrated polyps and two large colon cancer cohorts, the Cancer Genome Atlas (TCGA; n = 153), and French national Cartes d'Identité des Tumeurs (CIT) program (n = 462), with enhanced gene annotation through natural language processing techniques of the existing medical corpus. We reproducibly associate higher expression of the ligand-receptor axis of TFF2 and CXCR4 with BRAF V600E-mutant colon cancer (P = 3.0 × 10(-3) and 0.077, respectively for TCGA; P = 3.0 × 10(-8) and 5.1 × 10(-7) for CIT). Given well-described oncogenic roles of TFF2 and CXCR4 in colon cancer, and availability of CXCR4 inhibitors for other clinical indications, this ligand-receptor axis may represent an actionable target for prevention and treatment of this molecular subtype of colorectal cancer.