Thomas J. Devers

Cloning and variation of ground state intestinal stem cells

Stem cells of the gastrointestinal tract, pancreas, liver and other columnar epithelia collectively resist cloning in their elemental states. Here we demonstrate the cloning and propagation of highly clonogenic, ‘ground state’ stem cells of the human intestine and colon. We show that derived stem-cell pedigrees sustain limited copy number and sequence variation despite extensive serial passaging and display exquisitely precise, cell-autonomous commitment to epithelial differentiation consistent with their origins along the intestinal tract. This developmentally patterned and epigenetically maintained commitment of stem cells is likely to enforce the functional specificity of the adult intestinal tract. Using clonally derived colonic epithelia, we show that toxins A or B of the enteric pathogen Clostridium difficile recapitulate the salient features of pseudomembranous colitis. The stability of the epigenetic commitment programs of these stem cells, coupled with their unlimited replicative expansion and maintained clonogenicity, suggests certain advantages for their use in disease modelling and regenerative medicine.

Nanoproteomic analysis of extracellular receptor kinase-1/2 post-translational activation in microdissected human hyperplastic colon lesions.

Oncogenic activation resulting in hyperproliferative lesions within the colonic mucosa has been identified in putative precancerous lesions, aberrant crypt foci (ACF). KRAS and BRAF mutation status was determined in 172 ACF identified in the colorectum of screening subjects by in situ high‐definition, magnifying chromoendoscopy. Lesions were stratified according to histology (serrated vs. distended). Due to their limiting size, however, it was not technically feasible to examine downstream signaling consequences of these oncogenic mutations. We have combined ultraviolet‐infrared (UV/IR) microdissection with an ultrasensitive nanofluidic proteomic immunoassay (NIA) to enable accurate quantification of posttranslational modifications to mitogen‐activated protein kinase (MAPK) in total protein lysates isolated from hyperproliferative crypts and adjacent normal mucosa. Using this approach, levels of singly and dually (activated) phosphorylated isoforms of extracellular receptor kinase(ERK)‐1 and ERK‐2 were quantified in samples containing as little as 16 ng of total protein recovered from <200 cells. ERK activation is responsible for observed hyperplasia found in these early lesions, but is not directly dependent on KRAS and/or BRAF mutation status. This study describes the novel use of a sensitive nanofluidic platform to measure oncogene‐driven proteomic changes in diminutive lesions and highlights the advantage of this approach over classical immunohistochemistry‐based analyses.

HD Chromoendoscopy Coupled with DNA Mass Spectrometry Profiling Identifies Somatic Mutations in Microdissected Human Proximal Aberrant Crypt Foci

Despite increased implementation of screening colonoscopy, interval cancers in the proximal colon remain a major public health concern. This fact underscores the limitations of current screening paradigms and the need for developing advanced endoscopic techniques. The density of aberrant crypt foci (ACF), the earliest identifiable mucosal abnormality, may serve as a surrogate marker for colon cancer risk, but has rarely been studied in the proximal colon. To this end, high-definition (HD) chromoendoscopy was conducted to define the relevance of ACF in the proximal colon. In addition, due to limited ACF size, the development of a combinatorial approach was required to maximize data acquisition obtained from individual biopsy samples. Proximal and distal ACF samples were characterized for a total of 105 mutations across 22 known tumor suppressor and proto-oncogenes using high-throughput Sequenom MassARRAY analysis. From this profiling, a discrete number of somatic mutations were identified, including APCR876* and FLT3I836M, as well as a deletion within the EGFR gene. Combined, these data highlight the significance of ACF within the context of colon cancer pathogenesis, particularly in the proximal colon. Implications: The identification of cancer-related mutations in commonly overlooked mucosal lesions underscores the preventive benefit of implementing advanced endoscopic screening to larger patient populations, particularly in the proximal colon. Visual Overview: http://mcr.aacrjournals.org/content/early/2014/05/22/1541-7786.MCR-13-0624/F1.large.jpg. Mol Cancer Res; 12(6); 823–9. ©2014 AACR.

Genome-wide DNA methylation profiling reveals cancer-associated changes within early colonic neoplasia

Colorectal cancer (CRC) is characterized by genome-wide alterations to DNA methylation that influence gene expression and genomic stability. Less is known about the extent to which methylation is disrupted in the earliest stages of CRC development. In this study, we have combined laser-capture microdissection with reduced representation bisulfite sequencing to identify cancer-associated DNA methylation changes in human aberrant crypt foci (ACF), the earliest putative precursor to CRC. Using this approach, methylation profiles have been generated for 10 KRAS-mutant ACF and 10 CRCs harboring a KRAS mutation, as well as matched samples of normal mucosa. Of 811 differentially methylated regions (DMRs) identified in ACF, 537 (66%) were hypermethylated and 274 (34%) were hypomethylated. DMRs located within intergenic regions were heavily enriched for AP-1 transcription factor binding sites and were frequently hypomethylated. Furthermore, gene ontology analysis demonstrated that DMRs associated with promoters were enriched for genes involved in intestinal development, including homeobox genes and targets of the Polycomb repressive complex 2. Consistent with their role in the earliest stages of colonic neoplasia, 75% of the loci harboring methylation changes in ACF were also altered in CRC samples, though the magnitude of change at these sites was lesser in ACF. Although aberrant promoter methylation was associated with altered gene expression in CRC, this was not the case in ACF, suggesting the insufficiency of methylation changes to modulate gene expression in early colonic neoplasia. Altogether, these data demonstrate that DNA methylation changes, including significant hypermethylation, occur more frequently in early colonic neoplasia than previously believed, and identify epigenomic features of ACF that may provide new targets for cancer chemoprevention or lead to the development of new biomarkers for CRC risk.

Distinct Transcriptional Changes and Epithelial–Stromal Interactions Are Altered in Early-Stage Colon Cancer Development

Although the progression of mutated colonic cells is dependent upon interactions between the initiated epithelium and surrounding stroma, the nature of these interactions is poorly understood. Here, the development of an ultrasensitive laser capture microdissection (LCM)/RNA-seq approach for studying the epithelial and stromal compartments of aberrant crypt foci (ACF) is described. ACF are the earliest identifiable preneoplastic lesion found within the human colon and are detected using high-definition endoscopy with contrast dye spray. The current analysis focused on the epithelium of ACF with somatic mutations to either KRAS, BRAF, or APC, and expression patterns compared with normal mucosa from each patient. By comparing gene expression patterns among groups, an increase in a number of proinflammatory NF-κB target genes was identified that was specific to ACF epithelium, including TIMP1, RELA, and RELB. Distinct transcriptional changes associated with each somatic mutation were observed and a subset of ACF display BRAFV600E-mediated senescence-associated transcriptome characterized by increased expression of CDKN2A. Finally, LCM-captured ACF-associated stroma was found to be transcriptionally distinct from normal-appearing stroma, with an upregulation of genes related to immune cell infiltration and fibroblast activation. Immunofluorescence confirmed increased CD3+ T cells within the stromal microenvironment of ACF and an abundance of activated fibroblasts. Collectively, these results provide new insight into the cellular interplay that occurs at the earliest stages of colonic neoplasia, highlighting the important role of NF-κB, activated stromal fibroblasts, and lymphocyte infiltration. Implications: Fibroblasts and immune cells in the stromal microenvironment play an important role during the earliest stages of colon carcinogenesis. Mol Cancer Res; 14(9); 795–804. ©2016 AACR.

Proximal Aberrant Crypt Foci Associate with Synchronous Neoplasia and Are Primed for Neoplastic Progression

Aberrant crypt foci (ACF) are the earliest morphologically identifiable lesion found within the human colon. Despite their relatively high frequency in the distal colon, few studies have examined the molecular characteristics of ACF within the proximal colon. In the following study, clinical participants (n = 184) were screened for ACF using high-definition chromoendoscopy with contrast dye-spray. Following pathologic confirmation, ACF biopsies were subjected to laser capture microdissection (LCM), and epithelial cells were evaluated for somatic mutations with a customized colorectal cancer mutation panel using DNA-mass spectrometry. Samples were further characterized for microsatellite instability (MSI). Logistic models were used to associate proximal ACF with synchronous (detected during the same procedure) neoplasia. Thirty-nine percent of participants had at least one histologically confirmed proximal ACF. Individuals with a proximal ACF were significantly more likely to present with a synchronous neoplasm (P = 0.001), and specifically, a proximal, tubular, or tubulovillous adenoma (multivariable OR = 2.69; 95% confidence interval, 1.12–6.47; P = 0.027). Proximal ACF were more likely to be dysplastic (52%) compared with distal ACF (13%; P < 0.0001). Somatic mutations to APC, BRAF, KRAS, NRAS, and ERBB2 were detected in 37% of proximal ACF. Hyperplastic ACF were more often MSI-high, but there were no differences in MSI status observed by colonic location. In summary, ACF are identified in the proximal colons of approximately 40% of individuals undergoing chromoendoscopy and more often in patients with synchronous proximal adenomas. Implications: This study provides the most complete set of data, to date, that ACF represent the earliest step in the adenoma–carcinoma sequence but remain below the detection limit of conventional endoscopy. Visual Overview: http//mcr.accrjournals.org/content/molcanres/16/3/486/F1.large.jpg. Mol Cancer Res; 16(3); 486–95. ©2017 AACR. Visual Overview

Targeted Transcriptional Profiling of Microdissected Biopsy Specimens Representing Early Colonic Neoplasia

Our incomplete understanding of the critical changes that accompany the earliest stages of tumor initiation provides a substantial hurdle for the development of novel intervention strategies for cancer prevention. Premalignant lesions are inherently difficult to characterize given their diminutive size, creating technical obstacles for accurate genetic profiling. Here, we describe an approach combining laser‐capture microdissection (LCM) with targeted RNA‐sequencing to study the transcriptional state of epithelial and stromal cells during the earliest detectable stage of human colorectal neoplasia, the aberrant crypt foci (ACF). We provide a robust and reproducible workflow for RNA isolation, library preparation, and expression profiling of laser‐captured cells from frozen OCT‐embedded tissue specimens. It is anticipated that the methodological approach outlined in this report will provide a framework for a broad range of microgenomics analyses that can be routinely applied to many other premalignant tissues. J. Cell. Biochem. 117: 2677–2681, 2016.

Abstract 3240: Proximal human aberrant crypt foci as surrogate markers of colorectal cancer risk

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Despite effective use of screening colonoscopy, colorectal cancer (CRC) remains the second leading cause of cancer-related deaths. ‘Interval’ cancers, or those occurring between screening colonoscopy procedures, are often found within the proximal (right) colon, underscoring the need for advanced screening approaches using high-definition chromoendoscopy. To better define the colonic mucosa at-risk, we have focused considerable effort on identification and molecular analysis of aberrant crypt foci (ACF), an early macroscopically detectable lesion commonly found in the distal colon. ACF may serve as a surrogate marker of cancer risk, but have rarely been studied within context of the right colon. We hypothesize that proximal ACF may associate with risk factors for CRC and act as a surrogate marker for CRC risk. While distal ACF are frequent (approximately 13 per patient) proximal ACF are not (<1 per patient). We report that subjects with at least one proximal ACF are significantly more likely to have higher ACF multiplicity (p = 0.0002) and more importantly, are more likely to harbor synchronous colonic neoplasia (OR=2.38 [1.24-4.59], p = 0.0087). Histologically, proximal ACF are more frequently dysplastic (41%) compared to distal colon ACF (8%). ACF (n=49) were analyzed for a panel of oncogenes and tumor suppressors using mass spectrometry (MS)-based genotyping (Sequenom). Among the 112 targets analyzed, after KRAS (20%) and BRAF (22%), APC mutations (12%) were most common and specifically associated with dysplasia. Due to their diminutive size and flat morphology, proximal ACF are almost certainly missed during routine colonoscopy. However, our findings suggest that the identification, removal and analysis of proximal ACF may be especially important during screening colonoscopy of high-risk individuals. Citation Format: David A. Drew, Matthew P. Hanley, Allen Mo, Gyuhyeong Goh, Nicole A. Horelik, Thomas J. Devers, Joel Levine, Richard G. Stevens, James J. Grady, Daniel W. Rosenberg. Proximal human aberrant crypt foci as surrogate markers of colorectal cancer risk. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3240. doi:10.1158/1538-7445.AM2014-3240

Endoscopic Features of Mucous Cap Polyps: A Way to Predict Serrated Polyps

Background/Aims The aims of the study were to identify whether a mucous-cap predicts the presence of serrated polyps, and to determine whether additional endoscopic findings predict the presence of a sessile serrated adenomas/polyp (SSA/P). Methods We analyzed 147 mucous-capped polyps with corresponding histology, during 2011–2014. Eight endoscopic features (presence of borders, elevation, rim of debris, location in the colon, size ≥10 mm, varicose vessels, nodularity, and alteration in mucosal folds) of mucous-capped polyps were examined to see if they can predict SSA/Ps. Results A total of 86% (n=126) of mucous-capped polyps were from the right sided serrated pathway (right-sided hyperplastic [n=83], SSA/Ps [n=43], traditional serrated adenoma [n=1]), 10% (n=15) were left-sided hyperplastic polyps, and 3% (n=5) were from the adenoma-carcinoma sequence. The presence of a mucous cap combined with varicose vessels was the only significant predictor for SSA/Ps. The other seven characteristics were not found to be statistically significant for SSA/Ps, although location in the colon and the presence of nodularity trended towards significance. Conclusions Our study suggests that mucous-capped polyps have high predictability for being a part of the serrated pathway. Gastroenterologists should be alert for a mucous-capped polyp with varicose veins, as these lesions have a higher risk of SSA/P.

Associations of dietary fat with risk of early neoplasia in the proximal colon in a population-based case–control study

PurposeExcess dietary fat consumption is strongly associated with the risk of colorectal cancer, but less is known about its role in the earliest stages of carcinogenesis, particularly within the proximal colon. In the following case–control study, we evaluated the relationship between the intake of dietary fats and the frequency of early proximal neoplasia [aberrant crypt foci (ACF) or polyps], detectable by high-definition colonoscopy with contrast dye-spray.MethodsAverage-risk screening individuals underwent a high-definition colonoscopy procedure as part of larger ongoing clinical study of precancerous lesions in the proximal colon. Dietary fat intake was assessed using the Block Brief Food Frequency Questionnaire, which estimates average dietary intake based on 70 food items. The diets of individuals with no endoscopically identifiable lesions (n = 36) were compared to those with either ACF or polyps detected in the proximal colon.ResultsIn multivariate analysis, high dietary intake of total polyunsaturated fatty acids (PUFAs) and intake of omega-6 and omega-3 fatty acids were positively associated with neoplastic lesions in the proximal colon. When comparing ACF and polyp groups separately, a positive association was observed for both proximal polyps (OR 2.28; CI 1.16–7.09) and ACF (OR 2.86; CI 1.16–7.09) for total PUFA intake. Furthermore, the prevalence of proximal ACF was increased with higher intake of omega-6 (OR 3.54; CI 1.32–9.47) and omega-3 fatty acids (OR 2.29; CI 1.02–5.13), although there was no discernible difference in the omega-6/omega-3 ratio.ConclusionsThese results suggest that dietary PUFAs may be positively associated with risk of early neoplasia in the proximal colon. This study provides further evidence that dietary PUFA composition may play an important role in altering the microenvironment within the human colon.