Differential pre-malignant programs and microenvironment chart distinct paths to malignancy in human colorectal polyps.

Colorectal cancers (CRCs) arise from precursor polyps whose cellular origins, molecular heterogeneity, and immunogenic potential may reveal diagnostic and therapeutic insights when analyzed at high resolution. We present a single-cell transcriptomic and imaging atlas of the two most common human colorectal polyps, conventional adenomas and serrated polyps, and their resulting CRC counterparts. Integrative analysis of 128 datasets from 62 participants reveals adenomas arise from WNT-driven expansion of stem cells, while serrated polyps derive from differentiated cells through gastric metaplasia. Metaplasia-associated damage is coupled to a cytotoxic immune microenvironment preceding hypermutation, driven partly by antigen-presentation differences associated with tumor cell-differentiation status. Microsatellite unstable CRCs contain distinct non-metaplastic regions where tumor cells acquire stem cell properties and cytotoxic immune cells are depleted. Our multi-omic atlas provides insights into malignant progression of colorectal polyps and their microenvironment, serving as a framework for precision surveillance and prevention of CRC.

A Cell-free DNA Blood-Based Test for Colorectal Cancer Screening.

BACKGROUND: Colorectal cancer is the third most diagnosed cancer in adults in the United States. Early detection could prevent more than 90% of colorectal cancer-related deaths, yet more than one third of the screening-eligible population is not up to date with screening despite multiple available tests. A blood-based test has the potential to improve screening adherence, detect colorectal cancer earlier, and reduce colorectal cancer-related mortality. METHODS: We assessed the performance characteristics of a cell-free DNA (cfDNA) blood-based test in a population eligible for colorectal cancer screening. The coprimary outcomes were sensitivity for colorectal cancer and specificity for advanced neoplasia (colorectal cancer or advanced precancerous lesions) relative to screening colonoscopy. The secondary outcome was sensitivity to detect advanced precancerous lesions. RESULTS: The clinical validation cohort included 10,258 persons, 7861 of whom met eligibility criteria and were evaluable. A total of 83.1% of the participants with colorectal cancer detected by colonoscopy had a positive cfDNA test and 16.9% had a negative test, which indicates a sensitivity of the cfDNA test for detection of colorectal cancer of 83.1% (95% confidence interval [CI], 72.2 to 90.3). Sensitivity for stage I, II, or III colorectal cancer was 87.5% (95% CI, 75.3 to 94.1), and sensitivity for advanced precancerous lesions was 13.2% (95% CI, 11.3 to 15.3). A total of 89.6% of the participants without any advanced colorectal neoplasia (colorectal cancer or advanced precancerous lesions) identified on colonoscopy had a negative cfDNA blood-based test, whereas 10.4% had a positive cfDNA blood-based test, which indicates a specificity for any advanced neoplasia of 89.6% (95% CI, 88.8 to 90.3). Specificity for negative colonoscopy (no colorectal cancer, advanced precancerous lesions, or nonadvanced precancerous lesions) was 89.9% (95% CI, 89.0 to 90.7). CONCLUSIONS: In an average-risk screening population, this cfDNA blood-based test had 83% sensitivity for colorectal cancer, 90% specificity for advanced neoplasia, and 13% sensitivity for advanced precancerous lesions. (Funded by Guardant Health; ECLIPSE ClinicalTrials.gov number, NCT04136002.).

Circulating tryptophan-kynurenine pathway metabolites are associated with all-cause mortality among patients with stage I-III colorectal cancer.

Alterations within the tryptophan-kynurenine metabolic pathway have been linked to the etiology of colorectal cancer (CRC), but the relevance of this pathway for prognostic outcomes in CRC patients needs further elucidation. Therefore, we investigated associations between circulating concentrations of tryptophan-kynurenine pathway metabolites and all-cause mortality among CRC patients. This study utilizes data from 2102 stage I-III CRC patients participating in six prospective cohorts involved in the international FOCUS Consortium. Preoperative circulating concentrations of tryptophan, kynurenine, kynurenic acid (KA), 3-hydroxykynurenine (HK), xanthurenic acid (XA), 3-hydroxyanthranilic acid (HAA), anthranilic acid (AA), picolinic acid (PA), and quinolinic acid (QA) were measured by liquid chromatography-tandem mass spectrometry. Using Cox proportional hazards regression, we examined associations of above-mentioned metabolites with all-cause mortality, adjusted for potential confounders. During a median follow-up of 3.2 years (interquartile range: 2.2-4.9), 290 patients (13.8%) deceased. Higher blood concentrations of tryptophan, XA, and PA were associated with a lower risk of all-cause mortality (per doubling in concentrations: tryptophan: HR = 0.56; 95%CI:0.41,0.76, XA: HR = 0.74; 95%CI:0.64,0.85, PA: HR = 0.76; 95%CI:0.64,0.92), while higher concentrations of HK and QA were associated with an increased risk of death (per doubling in concentrations: HK: HR = 1.80; 95%CI:1.47,2.21, QA: HR = 1.31; 95%CI:1.05,1.63). A higher kynurenine-to-tryptophan ratio, a marker of cell-mediated immune activation, was associated with an increased risk of death (per doubling: HR = 2.07; 95%CI:1.52,2.83). In conclusion, tryptophan-kynurenine pathway metabolites may be prognostic markers of survival in CRC patients.

Significance of Crypt Atypia in Barrett's Esophagus: A Clinical, Molecular, and Outcome Study.

BACKGROUND & AIMS: The aim of this study was to characterize baseline morphologic features of crypts in nondysplastic Barrett's esophagus and correlate them with DNA content abnormalities and risk of progression to high-grade dysplasia (HGD) or esophageal adenocarcinoma (EAC). METHODS: The morphologic features of nondysplastic crypts in baseline biopsy specimens from 212 BE patients (2956 biopsy specimens) were graded histologically using a 4-point scale (crypt atypia levels, 0-3). DNA content abnormalities were detected using flow cytometry. RESULTS: In patients who had dysplasia in their baseline biopsy specimens, dysplasia was associated significantly with increasing grades of crypt atypia in the background nondysplastic Barrett's esophagus (P < .001). In a subset of patients without dysplasia at baseline (N = 149), a higher grade of crypt atypia was associated with longer Barrett's esophagus segment length (5.5 vs 3.3 cm; P = .0095), and a higher percentage of cells with 4N DNA content (3.67 ± 1.27 vs 2.93 ± 1.22; P = .018). Crypt atypia was associated with the development of any neoplasia (low-grade dysplasia and HGD/EAC). Although no significant association was noted between the grade of crypt atypia and increased 4N, aneuploidy, or progression to HGD/EAC, only patients with grade 2 or 3 crypt atypia showed increased 4N, aneuploidy, or progression to HGD/EAC. CONCLUSIONS: Patients with Barrett's esophagus likely develop dysplasia via a progressive increase in the level of crypt atypia before the onset of dysplasia, and these changes may reflect some alteration of DNA content.

Natural History of Colorectal Polyps Undergoing Longitudinal in Vivo CT Colonography Surveillance.

Background The natural history of colorectal polyps is not well characterized due to clinical standards of care and other practical constraints limiting in vivo longitudinal surveillance. Established CT colonography (CTC) clinical screening protocols allow surveillance of small (6-9 mm) polyps. Purpose To assess the natural history of colorectal polyps followed with CTC in a clinical screening program, with histopathologic correlation for resected polyps. Materials and Methods In this retrospective study, CTC was used to longitudinally monitor small colorectal polyps in asymptomatic adult patients from April 1, 2004, to August 31, 2020. All patients underwent at least two CTC examinations. Polyp growth patterns across multiple time points were analyzed, with histopathologic context for resected polyps. Regression analysis was performed to evaluate predictors of advanced histopathology. Results In this study of 475 asymptomatic adult patients (mean age, 56.9 years ± 6.7 [SD]; 263 men), 639 unique polyps (mean initial diameter, 6.3 mm; volume, 50.2 mm3) were followed for a mean of 5.1 years ± 2.9. Of these 639 polyps, 398 (62.3%) underwent resection and histopathologic evaluation, and 41 (6.4%) proved to be histopathologically advanced (adenocarcinoma, high-grade dysplasia, or villous content), including two cancers and 38 tubulovillous adenomas. Advanced polyps showed mean volume growth of +178% per year (752% per year for adenocarcinomas) compared with +33% per year for nonadvanced polyps and -3% per year for unresected, unretrieved, or resolved polyps (P < .001). In addition, 90% of histologically advanced polyps achieved a volume of 100 mm3 and/or volume growth rate of 100% per year, compared with 29% of nonadvanced and 16% of unresected or resolved polyps (P < .001). Polyp volume-to-diameter ratio was also significantly greater for advanced polyps. For polyps observed at three or more time points, most advanced polyps demonstrated an initial slower growth interval, followed by a period of more rapid growth. Conclusion Small colorectal polyps ultimately proving to be histopathologically advanced neoplasms demonstrated substantially faster growth and attained greater overall size compared with nonadvanced polyps. Clinical trial registration no. NCT00204867 © RSNA, 2024 Supplemental material is available for this article. See also the editorial by Dachman in this issue.

Risk factors and health behaviors associated with loneliness among cancer survivors during the COVID-19 pandemic.

Loneliness may exacerbate poor health outcomes particularly among cancer survivors during the COVID-19 pandemic. Little is known about the risk factors of loneliness among cancer survivors. We evaluated the risk factors of loneliness in the context of COVID-19 pandemic-related prevention behaviors and lifestyle/psychosocial factors among cancer survivors. Cancer survivors (n = 1471) seen at Huntsman Cancer Institute completed a survey between August-September 2020 evaluating health behaviors, medical care, and psychosocial factors including loneliness during COVID-19 pandemic. Participants were classified into two groups: 'lonely' (sometimes, usually, or always felt lonely in past month) and 'non-lonely' (never or rarely felt lonely in past month). 33% of cancer survivors reported feeling lonely in the past month. Multivariable logistic regression showed female sex, not living with a spouse/partner, poor health status, COVID-19 pandemic-associated lifestyle factors including increased alcohol consumption and marijuana/CBD oil use, and psychosocial stressors such as disruptions in daily life, less social interaction, and higher perceived stress and financial stress were associated with feeling lonely as compared to being non-lonely (all p < 0.05). A significant proportion of participants reported loneliness, which is a serious health risk among vulnerable populations, particularly cancer survivors. Modifiable risk factors such as unhealthy lifestyle behaviors and psychosocial stress were associated with loneliness. These results highlight the need to screen for unhealthy lifestyle factors and psychosocial stressors to identify cancer survivors at increased risk of loneliness and to develop effective management strategies.

Growth rates and histopathological outcomes of small (6-9 mm) colorectal polyps based on CT colonography surveillance and endoscopic removal.

BACKGROUND AND AIMS: The natural history of small polyps is not well established and rests on limited evidence from barium enema studies decades ago. Patients with one or two small polyps (6-9 mm) at screening CT colonography (CTC) are offered CTC surveillance at 3 years but may elect immediate colonoscopy. This practice allows direct observation of the growth of subcentimetre polyps, with histopathological correlation in patients undergoing subsequent polypectomy. DESIGN: Of 11 165 asymptomatic patients screened by CTC over a period of 16.4 years, 1067 had one or two 6-9 mm polyps detected (with no polyps ≥10 mm). Of these, 314 (mean age, 57.4 years; M:F, 141:173; 375 total polyps) elected immediate colonoscopic polypectomy, and 382 (mean age 57.0 years; M:F, 217:165; 481 total polyps) elected CTC surveillance over a mean of 4.7 years. Volumetric polyp growth was analysed, with histopathological correlation for resected polyps. Polyp growth and regression were defined as volume change of ±20% per year, with rapid growth defined as +100% per year (annual volume doubling). Regression analysis was performed to evaluate predictors of advanced histology, defined as the presence of cancer, high-grade dysplasia (HGD) or villous components. RESULTS: Of the 314 patients who underwent immediate polypectomy, 67.8% (213/314) harboured adenomas, 2.2% (7/314) with advanced histology; no polyps contained cancer or HGD. Of 382 patients who underwent CTC surveillance, 24.9% (95/382) had polyps that grew, while 62.0% (237/382) remained stable and 13.1% (50/382) regressed in size. Of the 58.6% (224/382) CTC surveillance patients who ultimately underwent colonoscopic resection, 87.1% (195/224) harboured adenomas, 12.9% (29/224) with advanced histology. Of CTC surveillance patients with growing polyps who underwent resection, 23.2% (19/82) harboured advanced histology vs 7.0% (10/142) with stable or regressing polyps (OR: 4.0; p<0.001), with even greater risk of advanced histology in those with rapid growth (63.6%, 14/22, OR: 25.4; p<0.001). Polyp growth, but not patient age/sex or polyp morphology/location were significant predictors of advanced histology. CONCLUSION: Small 6-9 mm polyps present overall low risk to patients, with polyp growth strongly associated with higher risk lesions. Most patients (75%) with small 6-9 mm polyps will see polyp stability or regression, with advanced histology seen in only 7%. The minority of patients (25%) with small polyps that do grow have a 3-fold increased risk of advanced histology.

Eflornithine plus Sulindac for Prevention of Progression in Familial Adenomatous Polyposis.

BACKGROUND: The efficacy and safety of combination therapy with eflornithine and sulindac, as compared with either drug alone, in delaying disease progression in patients with familial adenomatous polyposis are unknown. METHODS: We evaluated the efficacy and safety of the combination of eflornithine and sulindac, as compared with either drug alone, in adults with familial adenomatous polyposis. The patients were stratified on the basis of anatomical site with the highest polyp burden and surgical status; the strata were precolectomy (shortest projected time to disease progression), rectal or ileal pouch polyposis after colectomy (longest projected time), and duodenal polyposis (intermediate projected time). The patients were then randomly assigned in a 1:1:1 ratio to receive 750 mg of eflornithine, 150 mg of sulindac, or both once daily for up to 48 months. The primary end point, assessed in a time-to-event analysis, was disease progression, defined as a composite of major surgery, endoscopic excision of advanced adenomas, diagnosis of high-grade dysplasia in the rectum or pouch, or progression of duodenal disease. RESULTS: A total of 171 patients underwent randomization. Disease progression occurred in 18 of 56 patients (32%) in the eflornithine-sulindac group, 22 of 58 (38%) in the sulindac group, and 23 of 57 (40%) in the eflornithine group, with a hazard ratio of 0.71 (95% confidence interval [CI], 0.39 to 1.32) for eflornithine-sulindac as compared with sulindac (P = 0.29) and 0.66 (95% CI, 0.36 to 1.24) for eflornithine-sulindac as compared with eflornithine. Among 37 precolectomy patients, the corresponding values in the treatment groups were 2 of 12 patients (17%), 6 of 13 (46%), and 5 of 12 (42%) (hazard ratios, 0.30 [95% CI, 0.07 to 1.32] and 0.20 [95% CI, 0.03 to 1.32]); among 34 patients with rectal or ileal pouch polyposis, the values were 4 of 11 patients (36%), 2 of 11 (18%), and 5 of 12 (42%) (hazard ratios, 2.03 [95% CI, 0.43 to 9.62] and 0.84 [95% CI, 0.24 to 2.90]); and among 100 patients with duodenal polyposis, the values were 12 of 33 patients (36%), 14 of 34 (41%), and 13 of 33 (39%) (hazard ratios, 0.73 [95% CI, 0.34 to 1.52] and 0.76 [95% CI, 0.35 to 1.64]). Adverse and serious adverse events were similar across the treatment groups. CONCLUSIONS: In this trial involving patients with familial adenomatous polyposis, the incidence of disease progression was not significantly lower with the combination of eflornithine and sulindac than with either drug alone. (Funded by Cancer Prevention Pharmaceuticals; ClinicalTrials.gov number, NCT01483144; EudraCT number, 2012-000427-41.).

Emerging Tests for Noninvasive Colorectal Cancer Screening.

Colorectal cancer (CRC) is among the most common cancers globally and a major cause of cancer-related deaths. The American Cancer Society estimates that CRC will kill 1 in 60 Americans, and CRC screening is recommended for all Americans ≥45 years of age. Current CRC screening methods are effective for preventing CRC and have been shown to reduce CRC-related mortality. However, none of the currently available tests is ideal, and many people are not compliant with screening recommendations. Novel screening tests based on advances in CRC molecular biology, genetics, and epigenetics, combined with developments in sequencing technologies and computational analytic methods, have been developed to address the shortcomings of current CRC screening tests. These emerging tests include blood-based assays that use plasma-derived circulating tumor DNA and serum proteins to detect early CRC and advanced adenomas, assays that use stool DNA or mRNA, and methods for profiling the gut microbiome. Here we review current screening modalities, and we discuss the principles behind the most promising emerging CRC screening tests and the data supporting their potential to be used in clinical practice.

Patients With Esophageal Adenocarcinoma With Prior Gastroesophageal Reflux Disease Symptoms Are Similar to Those Without Gastroesophageal Reflux Disease: A Cross-Sectional Study.

INTRODUCTION: A substantial proportion of patients with esophageal adenocarcinoma (EAC) do not report gastroesophageal reflux disease (GERD) symptoms. This study aimed to compare the risk factor profiles and cancer stage at presentation of patients with EAC with and without prior GERD. METHODS: In this retrospective cross-sectional study, patients with EAC were divided into 2 cohorts: (i) EAC with prior GERD: patients who reported typical GERD symptoms (heartburn or regurgitation) ≥1 year before cancer diagnosis and (ii) EAC without prior GERD: patients who did not report prior GERD symptoms or reported symptoms within 1 year of their cancer diagnosis. Baseline demographics, risk factors, and cancer stage at presentation were compared between the 2 cohorts. In addition, the distribution of patients based on numbers of BE/EAC-associated risk factors (1, 2, 3, 4, and 5 or more) was examined in the symptomatic and asymptomatic cohorts. RESULTS: Over 13 years, 388 patients with EAC with prior GERD and 245 patients with EAC without prior GERD were recruited. Both groups had similar baseline demographics and risk factors, but patients with EAC with prior GERD were more likely to have a history of BE. Asymptomatic patients had more advanced disease. Patients with 3 or more BE/EAC-related risk factors formed the largest proportion of patients in both the symptomatic and asymptomatic cohorts. DISCUSSION: Patients with EAC with and without prior GERD symptoms are phenotypically similar, suggesting that BE screening efforts to prevent or detect early EAC should not be restricted to just those with GERD.

Artificial Intelligence-Triaged 3-Dimensional Pathology to Improve Detection of Esophageal Neoplasia While Reducing Pathologist Workloads.

Early detection of esophageal neoplasia via evaluation of endoscopic surveillance biopsies is the key to maximizing survival for patients with Barrett's esophagus, but it is hampered by the sampling limitations of conventional slide-based histopathology. Comprehensive evaluation of whole biopsies with 3-dimensional (3D) pathology may improve early detection of malignancies, but large 3D pathology data sets are tedious for pathologists to analyze. Here, we present a deep learning-based method to automatically identify the most critical 2-dimensional (2D) image sections within 3D pathology data sets for pathologists to review. Our method first generates a 3D heatmap of neoplastic risk for each biopsy, then classifies all 2D image sections within the 3D data set in order of neoplastic risk. In a clinical validation study, we diagnose esophageal biopsies with artificial intelligence-triaged 3D pathology (3 images per biopsy) vs standard slide-based histopathology (16 images per biopsy) and show that our method improves detection sensitivity while reducing pathologist workloads.

Laparoscopic prophylactic total gastrectomy with limited lymphadenectomy for CDH1 gene carriers.

BACKGROUND: CDH1 gene mutations are the leading etiology of hereditary diffuse gastric cancer with cumulative lifetime risk ranging up to 83%. Prophylactic total gastrectomy (PTG) is, therefore, recommended for CDH1 carriers. A laparoscopic approach may reduce operative risk versus an open operation, thus leading more patients with CDH1 mutations to pursue PTG prior to cancer development. However, more experience and oncologic outcome data are needed for a laparoscopic approach and indicated lymphadenectomy. METHODS: A retrospective descriptive cohort study of adult patients with CDH1 mutations who underwent laparoscopic PTG with D1 lymphadenectomy between 2012 and 2022 was conducted at a single institution. All patients had preoperative EGD screening, and those with visible tumor lesions on surveillance EGD were excluded and not considered prophylactic. Demographics, family history, pathology, and operative course were obtained. Outcomes included complications, readmission, and postoperative weight change. RESULTS: Among 23 patients, median age was 48 years (IQR 37, 53) and 15 (65%) were female. Family history for gastric and/or lobular breast cancer was present in 22 (96%) patients. The median [IQR] time from positive genetic testing to PTG was 347 days [140, 625]. Pathologic evaluation showed five (22%) patients with foci of gastric cancer on pre-operative EGD biopsies, 10 (44%) in resected stomach specimens. All lymph nodes were negative. To address early postoperative complications, EJ anastomotic technique changed from EEA to GIA over the course of the study and feeding jejunostomy was no longer placed during PTG with minimal change in postoperative weight loss. CONCLUSIONS: This is the largest series, spanning 10 years at a single institution, dedicated solely to a laparoscopic approach for risk-reducing PTG. A laparoscopic approach with limited lymphadenectomy resulted in acceptable surgical and oncologic outcomes. Despite no visible cancer, over half of our patients had foci of early gastric cancer. Therefore, CDH1 carriers should consider laparoscopic PTG.

Epigenetic Alterations in the Gastrointestinal Tract: Current and Emerging Use for Biomarkers of Cancer.

Colorectal cancer, liver cancer, stomach cancer, pancreatic cancer, and esophageal cancer are leading causes of cancer-related deaths worldwide. A fundamental trait of virtually all gastrointestinal cancers is genomic and epigenomic DNA alterations. Cancer cells acquire genetic and epigenetic alterations that drive the initiation and progression of the cancers by altering the molecular and cell biological processes of the cells. These alterations, as well as other host and microenvironment factors, ultimately mediate the clinical behavior of the precancers and cancers and can be used as biomarkers for cancer risk determination, early detection of cancer and precancer, determination of the prognosis of cancer and prediction of the response to therapy. Epigenetic alterations have emerged as one of most robust classes of biomarkers and are the basis for a growing number of clinical tests for cancer screening and surveillance.

Epigenetic silencing and tumor suppressor gene of HAND2 by targeting ERK signaling in colorectal cancer.

BACKGROUND: The screening biomarkers for early detection of colorectal cancer (CRC) is lacking. The aim is to identify epigenetic silenced genes and clarify its roles and underlying mechanism in CRC. We conducted integrative analyses of epigenome-wide Human Methylation 450 K arrays and transcriptome to screen out candidate epigenetic driver genes with transcription silencing. Methylated silencing HAND2 were identified and verified in large CRC cohort. The mechanism of HAND2 expression by promoter inhibition were clarified both in vitro and vivo assays. Cell biofunctional roles of HAND2 methylation was investigated in CRC cells. HAND2 reconstitution were constructed by lentivirus plasmid and tumor xenograft model of HAND2 were built subcutaneously. Genomic mRNA analysis by RNA-sequencing and subsequent GSEA analysis were performed to identify potential target of HAND2 and qPCR/WB was conducted to identify the results. RESULTS: We firstly reported high frequency of HAND2 methylation in promoter in CRC and hypermethylation was negatively correlated with expression silencing and leaded to poor survival in several CRC cohort patients. 5-Aza treatment to demethylated HAND2 could revert its expression in CRC cells. Functionally, HAND2 reconstitution can inhibit cell proliferation, invasion and migration in vitro. In tumor xenograft, HAND2 reconstruction significantly repressed tumor growth when compared to control vector. Thousands of aberrant expressed genes were observed in the heatmap of RNA-sequencing data. HAND2 reconstitution could bind to ERK and reduce its phosphorylation by CoIP assay. These above results showed HAND2 reconstitution perturbed the activation of MAPK/ERK signaling by reduction of ERK phosphorylation. CONCLUSIONS: HAND2 is one tumor suppressor by targeting ERK signaling and one potential epigenetic driver gene in CRC. Video Abstract.

AGA White Paper: Roadmap for the Future of Colorectal Cancer Screening in the United States.

The American Gastroenterological Association's Center for Gastrointestinal Innovation and Technology convened a consensus conference in December 2018, entitled, "Colorectal Cancer Screening and Surveillance: Role of Emerging Technology and Innovation to Improve Outcomes." The goal of the conference, which attracted more than 60 experts in screening and related disciplines, including the authors, was to envision a future in which colorectal cancer (CRC) screening and surveillance are optimized, and to identify barriers to achieving that future. This White Paper originates from that meeting and delineates the priorities and steps needed to improve CRC outcomes, with the goal of minimizing CRC morbidity and mortality. A one-size-fits-all approach to CRC screening has not and is unlikely to result in increased screening uptake or desired outcomes owing to barriers stemming from behavioral, cultural, and socioeconomic causes, especially when combined with inefficiencies in deployment of screening technologies. Overcoming these barriers will require the following: efficient utilization of multiple screening modalities to achieve increased uptake; continued development of noninvasive screening tests, with iterative reassessments of how best to integrate new technologies; and improved personal risk assessment to better risk-stratify patients for appropriate screening testing paradigms.

Screen for reactivation of MeCP2 on the inactive X chromosome identifies the BMP/TGF-ß superfamily as a regulator of XIST expression.

Rett syndrome (RS) is a debilitating neurological disorder affecting mostly girls with heterozygous mutations in the gene encoding the methyl-CpG-binding protein MeCP2 on the X chromosome. Because restoration of MeCP2 expression in a mouse model reverses neurologic deficits in adult animals, reactivation of the wild-type copy of MeCP2 on the inactive X chromosome (Xi) presents a therapeutic opportunity in RS. To identify genes involved in MeCP2 silencing, we screened a library of 60,000 shRNAs using a cell line with a MeCP2 reporter on the Xi and found 30 genes clustered in seven functional groups. More than half encoded proteins with known enzymatic activity, and six were members of the bone morphogenetic protein (BMP)/TGF-ß pathway. shRNAs directed against each of these six genes down-regulated X-inactive specific transcript (XIST), a key player in X-chromosome inactivation that encodes an RNA that coats the silent X chromosome, and modulation of regulators of this pathway both in cell culture and in mice demonstrated robust regulation of XIST. Moreover, we show that Rnf12, an X-encoded ubiquitin ligase important for initiation of X-chromosome inactivation and XIST transcription in ES cells, also plays a role in maintenance of the inactive state through regulation of BMP/TGF-ß signaling. Our results identify pharmacologically suitable targets for reactivation of MeCP2 on the Xi and a genetic circuitry that maintains XIST expression and X-chromosome inactivation in differentiated cells.

Subtypes of Barrett's oesophagus and oesophageal adenocarcinoma based on genome-wide methylation analysis.

OBJECTIVE: To identify and characterise DNA methylation subtypes in oesophageal adenocarcinoma (EAC) and its precursor Barrett's oesophagus (BE). DESIGN: We performed genome-wide DNA methylation profiling on samples of non-dysplastic BE from cancer-free patients (n=59), EAC (n=23), normal squamous oesophagus (n=33) and normal fundus (n=9), and identified methylation subtypes using a recursively partitioned mixture model. We assessed genomic alterations for 9 BE and 22 EAC samples with massively parallel sequencing of 243 EAC-associated genes, and we conducted integrative analyses with transcriptome data to identify epigenetically repressed genes. We also carried out in vitro experiments treating EAC cell lines with 5-Aza-2'-Deoxycytidine (5-Aza-dC), short hairpin RNA knockdown and anticancer therapies. RESULTS: We identified and validated four methylation subtypes of EAC and BE. The high methylator subtype (HM) of EAC had the greatest number of activating events in ERBB2 (p<0.05, Student's t-test) and the highest global mutation load (p<0.05, Fisher's exact test). PTPN13 was silenced by aberrant methylation in the HM subtype preferentially and in 57% of EACs overall. In EAC cell lines, 5-Aza-dC treatment restored PTPN13 expression and significantly decreased its promoter methylation in HM cell lines (p<0.05, Welch's t-test). Inhibition of PTPN13 expression in the SK-GT-4 EAC cell line promoted proliferation, colony formation and migration, and increased phosphorylation in ERBB2/EGFR/Src kinase pathways. Finally, EAC cell lines showed subtype-specific responses to topotecan, SN-38 and palbociclib treatment. CONCLUSIONS: We identified and characterised methylator subtypes in BE and EAC. We further demonstrated the biological and clinical relevance of EAC methylator subtypes, which may ultimately help guide clinical management of patients with EAC.

Single cell lineage tracing reveals a role for TgfßR2 in intestinal stem cell dynamics and differentiation.

Intestinal stem cells (ISCs) are maintained by a niche mechanism, in which multiple ISCs undergo differential fates where a single ISC clone ultimately occupies the niche. Importantly, mutations continually accumulate within ISCs creating a potential competitive niche environment. Here we use single cell lineage tracing following stochastic transforming growth factor ß receptor 2 (TgfßR2) mutation to show cell autonomous effects of TgfßR2 loss on ISC clonal dynamics and differentiation. Specifically, TgfßR2 mutation in ISCs increased clone survival while lengthening times to monoclonality, suggesting that Tgfß signaling controls both ISC clone extinction and expansion, independent of proliferation. In addition, TgfßR2 loss in vivo reduced crypt fission, irradiation-induced crypt regeneration, and differentiation toward Paneth cells. Finally, altered Tgfß signaling in cultured mouse and human enteroids supports further the in vivo data and reveals a critical role for Tgfß signaling in generating precursor secretory cells. Overall, our data reveal a key role for Tgfß signaling in regulating ISCs clonal dynamics and differentiation, with implications for cancer, tissue regeneration, and inflammation.

Discovery of methylated circulating DNA biomarkers for comprehensive non-invasive monitoring of treatment response in metastatic colorectal cancer.

OBJECTIVE: Mutations in cell-free circulating DNA (cfDNA) have been studied for tracking disease relapse in colorectal cancer (CRC). This approach requires personalised assay design due to the lack of universally mutated genes. In contrast, early methylation alterations are restricted to defined genomic loci allowing comprehensive assay design for population studies. Our objective was to identify cancer-specific methylated biomarkers which could be measured longitudinally in cfDNA (liquid biopsy) to monitor therapeutic outcome in patients with metastatic CRC (mCRC). DESIGN: Genome-wide methylation microarrays of CRC cell lines (n=149) identified five cancer-specific methylated loci (EYA4, GRIA4, ITGA4, MAP3K14-AS1, MSC). Digital PCR assays were employed to measure methylation of these genes in tumour tissue DNA (n=82) and cfDNA from patients with mCRC (n=182). Plasma longitudinal assessment was performed in a patient subset treated with chemotherapy or targeted therapy. RESULTS: Methylation in at least one marker was detected in all tumour tissue samples and in 156 mCRC patient cfDNA samples (85.7%). Plasma marker prevalence was 71.4% for EYA4, 68.5% for GRIA4, 69.7% for ITGA4, 69.1% for MAP3K14-AS1% and 65.1% for MSC. Dynamics of methylation markers was not affected by treatment type and correlated with objective tumour response and progression-free survival. CONCLUSION: This five-gene methylation panel can be used to circumvent the absence of patient-specific mutations for monitoring tumour burden dynamics in liquid biopsy under different therapeutic regimens. This method might be proposed for assessing pharmacodynamics in clinical trials or when conventional imaging has limitations.