The GenoVA study: Equitable implementation of a pragmatic randomized trial of polygenic-risk scoring in primary care.

Polygenic risk scores (PRSs) hold promise for disease risk assessment and prevention. The Genomic Medicine at Veterans Affairs (GenoVA) Study is addressing three main challenges to the clinical implementation of PRSs in preventive care: defining and determining their clinical utility, implementing them in time-constrained primary care settings, and countering their potential to exacerbate healthcare disparities. The study processes used to test patients, report their PRS results to them and their primary care providers (PCPs), and promote the use of those results in clinical decision-making are modeled on common practices in primary care. The following diseases were chosen for their prevalence and familiarity to PCPs: coronary artery disease; type 2 diabetes; atrial fibrillation; and breast, colorectal, and prostate cancers. A randomized clinical trial (RCT) design and primary outcome of time-to-new-diagnosis of a target disease bring methodological rigor to the question of the clinical utility of PRS implementation. The study's pragmatic RCT design enhances its relevance to how PRS might reasonably be implemented in primary care. Steps the study has taken to promote health equity include the thoughtful handling of genetic ancestry in PRS construction and reporting and enhanced recruitment strategies to address underrepresentation in research participation. To date, enhanced recruitment efforts have been both necessary and successful: participants of underrepresented race and ethnicity groups have been less likely to enroll in the study than expected but ultimately achieved proportional representation through targeted efforts. The GenoVA Study experience to date offers insights for evaluating the clinical utility of equitable PRS implementation in adult primary care.

Quantitative p53 immunostaining aids in the detection of prevalent dysplasia.

AIMS: The lack of accepted scoring criteria has precluded the use of p53 in routine practice. We evaluate the utility of automated quantitative p53 analysis in risk stratifying Barrett's oesophagus (BE) patients using non-dysplastic BE (NDBE) biopsies in a multicentric cohort of BE progressor (P) and non-progressor (NP) patients. METHODS: NDBE biopsies prior to the diagnosis of advanced neoplasia from 75 BE-P, and index and last surveillance biopsies from 148 BE-NP were stained for p53, and scored digitally as 1+, 2+ and 3+. A secondary cohort of 30 BE-P was evaluated. RESULTS: Compared with BE-NP, BE-P was predominantly men (p=0.001), ≥55 years of age (p=0.008), with longer BE segments (71% vs 33%; p<0.001). The mean number of 3+p53 positive cells and 3+ positive glands were significantly more in BE-P versus BE-NP NDBE biopsies (175 vs 9.7, p<0.001; 9.8 vs 0.1; p<0.001, respectively). At a cut-off of ≥10 p53 (3+) positive cells, the sensitivity and specificity of the assay to identify BE-P were 39% and 93%. On multivariate analysis, scoring p53 in NDBE biopsies, age, gender and length of BE were significantly associated with neoplastic progression. 54% of patients classified as prevalent dysplasia showed an abnormal p53 immunohistochemical stain. These findings were validated in the secondary cohort. CONCLUSIONS: Automated p53 analysis in NDBE biopsies serves as a promising tool for assessing BE neoplastic progression and risk stratification. Our study highlights the practical applicability of p53 assay to routine surveillance practice and its ability to detect prevalent dysplasia.

Methylated Septin9 (mSEPT9): A promising blood-based biomarker for the detection and screening of early-onset colorectal cancer.

Early-onset colorectal cancer (EOCRC), defined as a diagnosis under age 50, is an emerging public health burden. As many of these individuals fall outside of screening guidelines, the development of a minimally invasive, accurate screening modality for this population is warranted. We evaluated the FDA-approved blood-based biomarker methylated Septin9 (mSEPT9) test as screening tool for EOCRC. EOCRC plasma, healthy plasma, and serum-free conditioned media from cancer cell lines was collected. Cell-free DNA (cfDNA) was isolated and bisulfite converted for use in the assay. mSEPT9 and ACTB measured using Epi proColon® V2.0. EOCRC plasma was collected at Massachusetts General Hospital (2005-2019) and controls were collected at the National Institutes of Health and by ZenBio Inc. (prior to 2019). Twenty-seven EOCRC cases, 48 healthy controls <50 years old, and 39 healthy controls ≥50 years old were included in this study. mSEPT9 was detected more frequently in EOCRC cases (88.9%) compared to healthy controls age <50 (4.2%) and ≥50 (15.4%), respectively (p<0.001). The sensitivity, specificity, positive predictive value, and negative predictive values of the mSEPT9 assay to detect EOCRC was 90.8% (95% CI: 84.7-96.9%), 88.9% (95% CI: 77.0-100.0%), 96.3% (95% CI: 92.3-100.0%), and 75.0% (95% CI 60.0-90.0%), respectively, compared to all healthy controls. mSEPT9 cfDNA level was an independent predictor of survival (p=0.02). mSEPT9 is a sensitive and specific biomarker for EOCRC detection. These results suggest that mSEPT9 may be useful in the detection of EOCRC, providing a minimally invasive method for screening in this growing population of CRC patients.

Incident Cancer Risk and Signatures Among Older MUTYH Carriers: Analysis of Population-Based and Genomic Cohorts.

MUTYH carriers have an increased colorectal cancer risk in case-control studies, with loss of heterozygosity (LOH) as the presumed mechanism. We evaluated cancer risk among carriers in a prospective, population-based cohort of older adults. In addition, we assessed if cancers from carriers demonstrated mutational signatures (G:C>T:A transversions) associated with early LOH. We calculated incident risk of cancer and colorectal cancer among 13,131 sequenced study participants of the ASPirin in Reducing Events in the Elderly cohort, stratified by sex and adjusting for age, smoking, alcohol use, BMI, polyp history, history of cancer, and aspirin use. MUTYH carriers were identified among 13,033 participants in The Cancer Genome Atlas and International Cancer Genome Consortium, and somatic signatures of cancers were analyzed. Male MUTYH carriers demonstrated an increased risk for overall cancer incidence [multivariable HR, 1.66; 95% confidence interval (CI), 1.03-2.68; P = 0.038] driven by increased colorectal cancer incidence (multivariable HR, 3.55; 95% CI, 1.42-8.78; P = 0.007), as opposed to extracolonic cancer incidence (multivariable HR, 1.40; 95% CI, 0.81-2.44; P = 0.229). Female carriers did not demonstrate increased risk of cancer, colorectal cancer, or extracolonic cancers. Analysis of mutation signatures from cancers of MUTYH carriers revealed no significant contribution toward early mutagenesis from widespread G:C>T:A transversions among gastrointestinal epithelial cancers. Among cancers from carriers, somatic transversions associated with base-excision repair deficiency are uncommon, suggestive of diverse mechanisms of carcinogenesis in carriers compared with those who inherit biallelic MUTYH mutations. PREVENTION RELEVANCE: Despite absence of loss of heterozygosity in colorectal cancers, elderly male MUTYH carriers appeared to be at increased of colorectal cancer.

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.

Verrucous esophageal carcinoma is a unique indolent subtype of squamous cell carcinoma: a systematic review and individual patient regression analysis.

BACKGROUND AND AIMS: Verrucous esophageal carcinoma (VEC) is a rare malignancy that presents a diagnostic challenge. We aim to characterize the clinical and genomic features, tumor behavior, and treatment outcomes of VEC to guide clinical practice. METHODS: We performed a systematic review of the literature and identified additional cases from Massachusetts General Hospital records and The Cancer Genome Atlas (TCGA). We obtained individual VEC patient data and analyzed publicly available clinicogenomic data from TCGA. We performed a regression analysis comparing cases of VEC to esophageal squamous cell carcinoma (ESCC) to identify factors influencing survival. RESULTS: A total of 135 patients were reported in 82 publications, and four unpublished cases from Massachusetts General Hospital (median age 65 years, 69% males, 48% smokers, 33% consumed alcohol). Symptoms were present at diagnosis in 95% of patients, most commonly dysphagia and weight loss. Median symptom onset to diagnosis time was 11.5 months with frequent misdiagnosis as Candida esophagitis. Among VEC cases with pathologic staging, lymph node metastases were rare (5%) compared to ESCC (40%). VEC was genomically characterized by enrichment of SMARCA4 missense mutations and a lack of pathogenic TP53 mutations. Despite its diagnostic elusiveness, in a multivariate regression analysis, VEC was detected at earlier stages (p = < 0.001) compared to ESCC, and advanced stage was the only significant factor affecting survival (p = 0.013). CONCLUSIONS: VEC is a rare, clinically and genomically distinct subtype of ESCC. Recognition and diagnosis of this lesion may allow the pursuit of curative and less morbid treatment strategies.

Prospective Evaluation of the Transparent, Elastomeric, Adaptable, Long-Lasting (TEAL) Respirator.

N95 filtering facepiece respirators (FFR) and surgical masks are essential in reducing airborne disease transmission, particularly during the COVID-19 pandemic. However, currently available FFR's and masks have major limitations, including masking facial features, waste, and integrity after decontamination. In a multi-institutional trial, we evaluated a transparent, elastomeric, adaptable, long-lasting (TEAL) respirator to evaluate success of qualitative fit test with user experience and biometric evaluation of temperature, respiratory rate, and fit of respirator using a novel sensor. There was a 100% successful fit test among participants, with feedback demonstrating excellent or good fit (90% of participants), breathability (77.5%), and filter exchange (95%). Biometric testing demonstrated significant differences between exhalation and inhalation pressures among a poorly fitting respirator, well-fitting respirator, and the occlusion of one filter of the respirator. We have designed and evaluated a transparent elastomeric respirator and a novel biometric feedback system that could be implemented in the hospital setting.

Genetic Variants in the Regulatory T cell-Related Pathway and Colorectal Cancer Prognosis.

BACKGROUND: High numbers of lymphocytes in tumor tissue, including T regulatory cells (Treg), have been associated with better colorectal cancer survival. Tregs, a subset of CD4+ T lymphocytes, are mediators of immunosuppression in cancer, and therefore variants in genes related to Treg differentiation and function could be associated with colorectal cancer prognosis. METHODS: In a prospective German cohort of 3,593 colorectal cancer patients, we assessed the association of 771 single-nucleotide polymorphisms (SNP) in 58 Treg-related genes with overall and colorectal cancer-specific survival using Cox regression models. Effect modification by microsatellite instability (MSI) status was also investigated because tumors with MSI show greater lymphocytic infiltration and have been associated with better prognosis. Replication of significant results was attempted in 2,047 colorectal cancer patients of the International Survival Analysis in Colorectal Cancer Consortium (ISACC). RESULTS: A significant association of the TGFBR3 SNP rs7524066 with more favorable colorectal cancer-specific survival [hazard ratio (HR) per minor allele: 0.83; 95% confidence interval (CI), 0.74-0.94; P value: 0.0033] was replicated in ISACC (HR: 0.82; 95% CI, 0.68-0.98; P value: 0.03). Suggestive evidence for association was found with two IL7 SNPs, rs16906568 and rs7845577. Thirteen SNPs with differential associations with overall survival according to MSI in the discovery analysis were not confirmed. CONCLUSIONS: Common genetic variation in the Treg pathway implicating genes such as TGFBR3 and IL7 was shown to be associated with prognosis of colorectal cancer patients. IMPACT: The implicated genes warrant further investigation.

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.

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.

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.

LGR5 in Barrett's Esophagus and its Utility in Predicting Patients at Increased Risk of Advanced Neoplasia.

INTRODUCTION: The expression of LGR5, a known stem cell marker, is poorly understood in Barrett's esophagus (BE) and related neoplasia. The aim of this study was to evaluate LGR5 in BE and related neoplasia and to evaluate its utility as a potential biomarker of progression to advanced neoplasia. METHODS: We evaluated total 137 patients, including 119 with BE and 18 with normal gastroesophageal mucosa for expression of LGR5 using RNA in situ hybridization; this also included 28 progressors and 30 nonprogressors. The LGR5 stain was evaluated using 1 qualitative and 2 quantitative parameters, using manual and automated platforms. RESULTS: Surface LGR5 expression was mainly seen in high-grade dysplasia (12/18) compared with low-grade dysplasia (1/8) and nondysplastic BE (0/17) (P < 0.0001). In contrast to nondysplastic BE, low- and high-grade dysplasia showed a higher percentage of mean number of LGR5-positive crypts per patient (P < 0.0001) and an increase in the mean number of LGR5 transcripts per cell (P < 0.0001). The mean percentage of LGR5-positive crypts per patient and the mean number of LGR5 transcripts per cell were also significantly higher in nondysplastic BE from progressor compared with nonprogressor (P < 0.0001, P = 0.014). The sensitivity and specificity of LGR5 for distinguishing progressor from nonprogressor were 50% and 87%, respectively. DISCUSSION: BE-related advanced neoplasia shows an expansion of the LGR5-positive cellular compartment, supporting its role as a stem cell marker in this disease. Quantitative LGR5 expression and surface epithelial reactivity are novel biomarkers of increased risk of progression to advanced neoplasia in BE.

DNA repair and cancer in colon and rectum: Novel players in genetic susceptibility.

Interindividual differences in DNA repair systems may play a role in modulating the individual risk of developing colorectal cancer. To better ascertain the role of DNA repair gene polymorphisms on colon and rectal cancer risk individually, we evaluated 15,419 single nucleotide polymorphisms (SNPs) within 185 DNA repair genes using GWAS data from the Colon Cancer Family Registry (CCFR) and the Genetics and Epidemiology of Colorectal Cancer Consortium (GECCO), which included 8,178 colon cancer, 2,936 rectum cancer cases and 14,659 controls. Rs1800734 (in MLH1 gene) was associated with colon cancer risk (p-value = 3.5 × 10-6 ) and rs2189517 (in RAD51B) with rectal cancer risk (p-value = 5.7 × 10-6 ). The results had statistical significance close to the Bonferroni corrected p-value of 5.8 × 10-6 . Ninety-four SNPs were significantly associated with colorectal cancer risk after Binomial Sequential Goodness of Fit (BSGoF) procedure and confirmed the relevance of DNA mismatch repair (MMR) and homologous recombination pathways for colon and rectum cancer, respectively. Defects in MMR genes are known to be crucial for familial form of colorectal cancer but our findings suggest that specific genetic variations in MLH1 are important also in the individual predisposition to sporadic colon cancer. Other SNPs associated with the risk of colon cancer (e.g., rs16906252 in MGMT) were found to affect mRNA expression levels in colon transverse and therefore working as possible cis-eQTL suggesting possible mechanisms of carcinogenesis.

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.

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.

A Mixed-Effects Model for Powerful Association Tests in Integrative Functional Genomics.

Genome-wide association studies (GWASs) have successfully identified thousands of genetic variants for many complex diseases; however, these variants explain only a small fraction of the heritability. Recently, genetic association studies that leverage external transcriptome data have received much attention and shown promise for discovering novel variants. One such approach, PrediXcan, is to use predicted gene expression through genetic regulation. However, there are limitations in this approach. The predicted gene expression may be biased, resulting from regularized regression applied to moderately sample-sized reference studies. Further, some variants can individually influence disease risk through alternative functional mechanisms besides expression. Thus, testing only the association of predicted gene expression as proposed in PrediXcan will potentially lose power. To tackle these challenges, we consider a unified mixed effects model that formulates the association of intermediate phenotypes such as imputed gene expression through fixed effects, while allowing residual effects of individual variants to be random. We consider a set-based score testing framework, MiST (mixed effects score test), and propose two data-driven combination approaches to jointly test for the fixed and random effects. We establish the asymptotic distributions, which enable rapid calculation of p values for genome-wide analyses, and provide p values for fixed and random effects separately to enhance interpretability over GWASs. Extensive simulations demonstrate that our approaches are more powerful than existing ones. We apply our approach to a large-scale GWAS of colorectal cancer and identify two genes, POU5F1B and ATF1, which would have otherwise been missed by PrediXcan, after adjusting for all known loci.