Clonal Transitions and Phenotypic Evolution in Barrett's Esophagus.

BACKGROUND & AIMS: Barrett's esophagus (BE) is a risk factor for esophageal adenocarcinoma but our understanding of how it evolves is poorly understood. We investigated BE gland phenotype distribution, the clonal nature of phenotypic change, and how phenotypic diversity plays a role in progression. METHODS: Using immunohistochemistry and histology, we analyzed the distribution and the diversity of gland phenotype between and within biopsy specimens from patients with nondysplastic BE and those who had progressed to dysplasia or had developed postesophagectomy BE. Clonal relationships were determined by the presence of shared mutations between distinct gland types using laser capture microdissection sequencing of the mitochondrial genome. RESULTS: We identified 5 different gland phenotypes in a cohort of 51 nondysplastic patients where biopsy specimens were taken at the same anatomic site (1.0-2.0 cm superior to the gastroesophageal junction. Here, we observed the same number of glands with 1 and 2 phenotypes, but 3 phenotypes were rare. We showed a common ancestor between parietal cell-containing, mature gastric (oxyntocardiac) and goblet cell-containing, intestinal (specialized) gland phenotypes. Similarly, we have shown a clonal relationship between cardiac-type glands and specialized and mature intestinal glands. Using the Shannon diversity index as a marker of gland diversity, we observed significantly increased phenotypic diversity in patients with BE adjacent to dysplasia and predysplasia compared to nondysplastic BE and postesophagectomy BE, suggesting that diversity develops over time. CONCLUSIONS: We showed that the range of BE phenotypes represents an evolutionary process and that changes in gland diversity may play a role in progression. Furthermore, we showed a common ancestry between gastric and intestinal-type glands in BE.

Reduced survival after upper gastrointestinal bleed endoscopy in the COVID-19 era is a secondary effect of the response to the global pandemic: a retrospective cohort study.

OBJECTIVE: The COVID-19 pandemic has placed increased strain on healthcare systems worldwide with enormous reorganisation undertaken to support 'COVID-centric' services. Non-COVID-19 admissions reduced secondary to public health measures to halt viral transmission. We aimed to understand the impact of the response to COVID-19 on the outcomes of upper gastrointestinal (UGI) bleeds. DESIGN/METHODS: A retrospective observational multicentre study comparing outcomes following endoscopy for UGI bleeds from 24 March 2020 to 20 April 2020 to the corresponding dates in 2019. The primary outcome was in-hospital survival at 30 days with secondary outcomes of major rebleeding within 30 days postprocedure and intervention at the time of endoscopy. RESULTS: 224 endoscopies for 203 patients with UGI bleeds were included within this study. 19 patients were diagnosed with COVID-19. There was a 44.4% reduction in the number of procedures performed between 2019 and 2020. Endoscopies performed for UGI bleeds in the COVID-19 era were associated with an adjusted reduced 30-day survival (OR 0.25, 95% CI 0.08-0.67). There was no increased risk of major rebleeding or interventions during this era. Patients with COVID-19 did not have reduced survival or increased complication rates. CONCLUSION: Endoscopy for UGI bleeds in the COVID-19 era is associated with reduced survival. No clear cause has been identified but we suspect that this is a secondary effect of the response to the COVID-19 pandemic. Urgent work is required to encourage the public to seek medical help if required and to optimise patient pathways to ensure that the best possible care is provided.

Distinct afferent innervation patterns within the human proximal and distal esophageal mucosa.

Little is known about the mucosal phenotype of the proximal human esophagus. There is evidence to suggest that the proximal esophagus is more sensitive to chemical and mechanical stimulation compared with the distal. This may have physiological relevance (e.g., in prevention of aspiration of gastroesophageal refluxate), but also pathological relevance (e.g., in reflux perception or dysphagia). Reasons for this increased sensitivity are unclear but may include impairment in mucosal barrier integrity or changes in sensory innervation. We assessed mucosal barrier integrity and afferent nerve distribution in the proximal and distal esophagus of healthy human volunteers. In 10 healthy volunteers baseline proximal and distal esophageal impedance was measured in vivo. Esophageal mucosal biopsies from the distal and proximal esophagus were taken, and baseline transepithelial electrical resistance (TER) was measured in Ussing chambers. Biopsies were examined immunohistochemically for presence and location of calcitonin gene-related peptide (CGRP)-immunoreactive nerve fibers. In a further four healthy volunteers we investigated for colocalization of CGRP and protein gene product (PGP) 9.5 immunoreactivity in nerve fibers. Baseline impedance was higher in the proximal than in the distal esophagus [2,936 Ω (SD578) vs. 2,229 Ω (SD821); P = 0.03], however, baseline TER was not significantly different between them. Mucosal CGRP-immunoreactive nerves were found in the epithelium of both proximal and distal esophagus, but were located more superficially in the proximal mucosa compared with the distal [11.5 (SD7) vs. 21.7 (SD5) cell layers from lumen, P = 0.002] 19% of proximal, and 10% of distal mucosal PGP-immunoreactive fibers colocalized with CGRP. PGP-immunoreactive fibers were also significantly closer to the luminal surface in the proximal compared with the distal esophagus (P < 0.001). We conclude that mucosal barrier integrity is similar in proximal and distal esophagus, but proximal mucosal afferent nerves are in a more superficial location. The enhanced sensitivity to reflux-evoked symptoms of the proximal esophagus most likely has an anatomical basis.

The clonal origins of dysplasia from intestinal metaplasia in the human stomach.

BACKGROUND & AIMS: Studies of the clonal architecture of gastric glands with intestinal metaplasia are important in our understanding of the progression from metaplasia to dysplasia. It is not clear if dysplasias are derived from intestinal metaplasia or how dysplasias expand. We investigated whether cells within a metaplastic gland share a common origin, whether glands clonally expand by fission, and determine if such metaplastic glands are genetically related to the associated dysplasia. We also examined the clonal architecture of entire dysplastic lesions and the genetic changes associated with progression within dysplasia. METHODS: Cytochrome c oxidase-deficient (CCO⁻) metaplastic glands were identified using a dual enzyme histochemical assay. Clonality was assessed by laser capture of multiple cells throughout CCO⁻ glands and polymerase chain reaction sequencing of the entire mitochondrial DNA (mtDNA) genome. Nuclear DNA abnormalities in individual glands were identified by laser capture microdissection polymerase chain reaction sequencing for mutation hot spots and microsatellite loss of heterozygosity analysis. RESULTS: Metaplastic glands were derived from the same clone-all lineages shared a common mtDNA mutation. Mutated glands were found in patches that had developed through gland fission. Metaplastic and dysplastic glands can be genetically related, indicating the clonal origin of dysplasia from metaplasia. Entire dysplastic fields contained a founder mutation from which multiple, distinct subclones developed. CONCLUSIONS: There is evidence for a distinct clonal evolution from metaplasia to dysplasia in the human stomach. By field cancerization, a single clone can expand to form an entire dysplastic lesion. Over time, this field appears to become genetically diverse, indicating that gastric cancer can arise from a subclone of the founder mutation.

Use of methylation patterns to determine expansion of stem cell clones in human colon tissue.

BACKGROUND & AIMS: It is a challenge to determine the dynamics of stem cells within human epithelial tissues such as colonic crypts. By tracking methylation patterns of nonexpressed genes, we have been able to determine how rapidly individual stem cells became dominant within a human colonic crypt. We also analyzed methylation patterns to study clonal expansion of entire crypts via crypt fission. METHODS: Colonic mucosa was obtained from 9 patients who received surgery for colorectal cancer. The methylation patterns of Cardiac-specific homeobox, Myoblast determination protein 1, and Biglycan were examined within clonal cell populations, comprising either part of, or multiple adjacent, normal human colonic crypts. Clonality was demonstrated by following cytochrome c oxidase-deficient (CCO⁻) cells that shared an identical somatic point mutation in mitochondrial DNA. RESULTS: Methylation pattern diversity among CCO⁻ clones that occupied only part of a crypt was proportional to clone size; this allowed us to determine rates of clonal expansion. Analysis indicated a slow rate of niche succession within the crypt. The 2 arms of bifurcating crypts had distinct methylation patterns, indicating that fission can disrupt epigenetic records of crypt ancestry. Adjacent clonal CCO⁻ crypts usually had methylation patterns as dissimilar to one another as methylation patterns of 2 unrelated crypts. Mathematical models indicated that stem cell dynamics and epigenetic drift could account for observed dissimilarities in methylation patterns. CONCLUSIONS: Methylation patterns can be analyzed to determine the rates of recent clonal expansion of stem cells, but determination of clonality over many decades is restricted by epigenetic drift. We developed a technique to follow changes in intestinal stem cell dynamics in human epithelial tissues that might be used to study premalignant disease.

Case report: achalasia-like dysmotility secondary to oesophageal involvement of sarcoidosis.

We present a case of a patient with sarcoidosis and who subsequently developed dysphagia for solids, and some difficulty in swallowing liquids. High-resolution manometry of the oesophagus showed absent peristalsis in the oesophageal body and incomplete relaxation of the lower oesophageal sphincter. The diagnosis of sarcoidosis with oesophageal involvement was made and treatment with prednisolone 30 mg OD initiated. The patient improved symptomatically and high-resolution manometry was repeated showing complete recovery of oesophageal peristalsis and a deeper relaxation of the lower oesophageal sphincter. This is thus the first description of high-resolution manometry in sarcoidosis-induced changes of the oesophagus and of the effect treatment has on these motility changes. Oesophageal involvement of sarcoidosis is extremely rare and only a few cases have been reported. The symptoms and manometric pattern of this disorder mimics that of achalasia. However, we show that treatment with prednisolone results in a completely disappearance of the symptoms of dysphagia and subsequently lead to a large improvement of oesophageal motility.

Esophageal adenocarcinoma in "mice and men": back to basics!

Adenocarcinoma related to Barrett's esophagus (BE) is increasing in the West faster than any other cancer. There are many potential chemopreventive agents as well as predictive biomarkers of cancer progression, but what is required is a robust high-throughput model in which to test hypotheses preclinically. The pathophysiology of metaplasia and cancer has been studied in 10 animal species. Though they have considerable genetic divergence, anatomical dissimilarity, and experimental flaws, they have provided some data to test in the clinic, especially relating to activation of common genetic pathways, role of hypergastrinemia, and duodenogastric reflux in cancer progression. In this regard, the human postesophagectomy model, which has a 30% recurrence of BE within 3 yr and a 5% recurrence of adenocarcinoma over 10 yr, is now being utilized to understand how human metaplasia occurs. Furthermore, improved clinical trial designs mean that more sophisticated questions can be addressed in man.

Mechanisms of field cancerization in the human stomach: the expansion and spread of mutated gastric stem cells.

BACKGROUND & AIMS: How mutations are established and spread through the human stomach is unclear because the clonal structure of gastric mucosal units is unknown. Here we investigate, using mitochondrial DNA (mtDNA) mutations as a marker of clonal expansion, the clonality of the gastric unit and show how mutations expand in normal mucosa and gastric mucosa showing intestinal metaplasia. This has important implications in gastric carcinogenesis. METHODS: Mutated units were identified by a histochemical method to detect activity of cytochrome c oxidase. Negative units were laser-capture microdissected, and mutations were identified by polymerase chain reaction sequencing. Differentiated epithelial cells were identified by immunohistochemistry for lineage markers. RESULTS: We show that mtDNA mutations establish themselves in stem cells within normal human gastric body units, and are passed on to all their differentiated progeny, thereby providing evidence for clonal conversion to a new stem cell-derived unit-monoclonal conversion, encompassing all gastric epithelial lineages. The presence of partially mutated units indicates that more than one stem cell is present in each unit. Mutated units can divide by fission to form patches, with each unit sharing an indentical, mutant mtDNA genotype. Furthermore, we show that intestinal metaplastic crypts are clonal, possess multiple stem cells, and that fission is a mechanism by which intestinal metaplasia spreads. CONCLUSIONS: These data show that human gastric body units are clonal, contain multiple multipotential stem cells, and provide definitive evidence for how mutations spread within the human stomach, and show how field cancerization develops.

Mechanisms of disease: from stem cells to colorectal cancer.

Over the past decade, the advances in our understanding of stem cell biology and the role of stem cells in diseases, such as colorectal cancer, have been remarkable. In particular, discoveries related to the control of stem cell proliferation and how dysregulation of proliferation leads to oncogenesis have been foremost. For intestinal stem cells, the WNT family of growth factors, and events such as the regulation of the nuclear localization of beta-catenin, seem to be central to normal homeostasis, and mutations in the components of these pathways seem to lead to the development of colorectal cancer. A paradigm of abnormal stem cell biology is illustrated by patients with familial adenomatous polyposis, who have mutations in the adenomatous polyposis coli gene. The wild-type protein encoded by this gene is important for the prevention of mass beta-catenin accumulation in the nucleus and the subsequent overtranscription of cell cycle proteins. This review discusses the basic mechanisms behind stem cell regulation in the gut and follows their role in the natural history of tumor progression.

Clonal expansion in the human gut: mitochondrial DNA mutations show us the way.

The mechanisms of how DNA mutations are fixed within the human gastrointestinal tract and how they spread are poorly understood and are hotly debated. It has been well documented that human colonic crypts are clonal units; one epithelial stem cell within the crypt becoming dominant and taking over the crypts' entire stem cell population--so called monoclonal conversion. Studies have revealed that crypts can exist as families and develop into patches. The questions have been how do such patches in the human colon develop? Does this have implications on how DNA mutations spread? We have previously shown that mitochondrial DNA (mtDNA) mutations, which result in the deficiency of cytochrome c oxidase, are established within a single colonic crypt stem cell, resulting in a crypt with a mixed phenotype. Over time that mutated stem cell can take over the entire stem cell population resulting in a wholly-mutated crypt. We have furthered this research by showing that entirely cytochrome c oxidase-deficient crypts are able to divide by a process called crypt fission, to form two cytochrome c oxidase-deficient daughter crypts, each sharing the exact parental mtDNA mutation. Furthermore, patches of these crypts also possess a founder mtDNA mutation suggesting that fission repeats itself to form patches, which increase in size with age. Here, we hypothesize that this can be expanded into other areas of the gastrointestinal tract, especially the stomach, where there is a paucity of data regarding clonality and the spread of DNA mutations. We ask if these mutated crypts expand at a different rate to wild type ones. We also discuss the implications for the spread of potential carcinogenic mutations within the gut.

Mitochondrial DNA mutations are established in human colonic stem cells, and mutated clones expand by crypt fission.

The understanding of the fixation of mutations within human tissues and their subsequent clonal expansion is a considerable problem, of which little is known. We have previously shown that nononcogenic mutations in the mitochondrial genome occur in one of a number of morphologically normal colonic crypt stem cells, the progeny of which later occupy the whole crypt. We propose that these wholly mutated crypts then clonally expand by crypt fission, where each crypt divides into two mutated daughter crypts. Here we show that (i) mutated crypts in the process of fission share the same mutated mitochondrial genotype not present in neighboring cytochrome c oxidase-positive crypts (the odds of this being a random event are >or=2.48 x 10(9):1); (ii) neighboring mutated crypts have the same genotype, which is different from adjacent cytochrome c oxidase-positive crypts; (iii) mutated crypts are clustered together throughout the colon; and (iv) patches of cytochrome c oxidase-deficient crypts increase in size with age. We thus demonstrate definitively that crypt fission is the mechanism by which mutations spread in the normal human colon. This has important implications for the biology of the normal adult human colon and possibly for the growth and spread of colorectal neoplasms.

Multiple organ engraftment by bone-marrow-derived myofibroblasts and fibroblasts in bone-marrow-transplanted mice.

Myofibroblasts are ubiquitous cells with features of both fibroblasts and smooth muscle cells. We suggest that the bone marrow can contribute to myofibroblast populations in a variety of tissues and that this is exacerbated by injury. To assess this, female mice were transplanted with male bone marrow and the male cells were tracked throughout the body and identified as myofibroblasts. Skin wounding and paracetamol administration were used to assess whether myofibroblast engraftment was modulated by damage. Following radiation injury, a proportion of myofibroblasts in the lung, stomach, esophagus, skin, kidney, and adrenal capsule were bone-marrow derived. In the lung, there was significantly greater engraftment following paracetamol administration (17% versus 41% p < 0.005). Bone-marrow-derived fibroblasts were also found. We suggest that bone marrow contributes to a circulating population of cells and, in the context of injury, these cells are recruited and contribute to tissue repair.

Bottom-up histogenesis of colorectal adenomas: origin in the monocryptal adenoma and initial expansion by crypt fission.

The adenoma:carcinoma sequence is well established. Understanding the molecular pathology of the adenoma is therefore important. There is great controversy within the field. The Vogelstein group champions the "top-down" theory (colorectal adenomas arise and grow across the mucosal surface and down into the crypts), whereas other studies, including our own, propose "bottom-up" spread. Serial sections of 40 small (<3 mm) sporadic colorectal adenomas were stained with H&E, MIB-1, and for beta-catenin. 10 early adenomas were Feulgen-stained and microdissected. We also examined the flat mucosa of three patients who had undergone colectomies for familial adenomatous polyposis (FAP) and specimens from a XO/XY individual with FAP, the latter using in situ hybridization for the Y chromosome. In the earliest sporadic adenomas, there were crypts entirely filled with adenomatous epithelium, which showed proliferative activity and nuclear localization of beta-catenin. There was a sharp cutoff between crypt epithelial cells showing nuclear beta-catenin and surface cells with membrane staining. In slightly larger lesions, adenomatous spread from above was seen. Microdissected adenomas showed multiple fission events, with proliferation distributed equally throughout. In FAP tissue, numerous isolated monocryptal adenomas, which were clonal in origin, were seen. Examination of adenomas in the XO/XY individual showed no instances of XY or XO adenomatous epithelium growing down into crypts of the other genotype. Both sporadic and FAP adenomas start as a unicryptal adenomas and grow initially by crypt fission--a bottom-up pattern. Later, in sporadic adenomas, there is evidence of growth down into adjacent crypts (top-down).

Plastic adult stem cells: will they graduate from the school of hard knocks?

Notwithstanding the fact that adult bone marrow cell engraftment to epithelial organs seems a somewhat uncommon event, there is no doubt it does occur, and under appropriate conditions of a strong and positive selection pressure these cells will expand clonally and make a significant contribution to tissue replacement. Likewise, bone-marrow-derived cells can be amplified in vitro and differentiated into a multitude of tissues. These in essence are the goals of regenerative medicine using any source of stem cells, be it embryonic or adult. Despite such irrefutable evidence of what is possible, a veritable chorus of detractors of adult stem cell plasticity has emerged, some doubting its very existence, motivated perhaps by more than a little self-interest. The issues that have led to this state of affairs have included the inability to reproduce certain widely quoted data, one case where the apparent transdifferentiation was due to contamination of the donor tissue with haematopoietic cells and, most notoriously, extrapolating from the behaviour of embryonic stem cells to suggest that adult bone marrow cells simply fuse with other cells and adopt their phenotype. While these issues need resolving, slamming this whole new field because not everything is crystal clear is not good science. The fact that a phenomenon is quite rare in no way mitigates against its very existence: asteroid collisions with the Earth are rare, but try telling the dinosaurs they do not occur! When such events do occur (transdifferentiation or collision), they certainly can make an impact.