Hepatocytes undergo punctuated expansion dynamics from a periportal stem cell niche in normal human liver.

BACKGROUND & AIMS: While normal human liver is thought to be generally quiescent, clonal hepatocyte expansions have been observed, though neither their cellular source nor their expansion dynamics have been determined. Knowing the hepatocyte cell of origin, and their subsequent dynamics and trajectory within the human liver will provide an important basis to understand disease-associated dysregulation. METHODS: Herein, we use in vivo lineage tracing and methylation sequence analysis to demonstrate normal human hepatocyte ancestry. We exploit next-generation mitochondrial sequencing to determine hepatocyte clonal expansion dynamics across spatially distinct areas of laser-captured, microdissected, clones, in tandem with computational modelling in morphologically normal human liver. RESULTS: Hepatocyte clones and rare SOX9+ hepatocyte progenitors commonly associate with portal tracts and we present evidence that clones can lineage-trace with cholangiocytes, indicating the presence of a bipotential common ancestor at this niche. Within clones, we demonstrate methylation CpG sequence diversity patterns indicative of periportal not pericentral ancestral origins, indicating a portal to central vein expansion trajectory. Using spatial analysis of mitochondrial DNA variants by next-generation sequencing coupled with mathematical modelling and Bayesian inference across the portal-central axis, we demonstrate that patterns of mitochondrial DNA variants reveal large numbers of spatially restricted mutations in conjunction with limited numbers of clonal mutations. CONCLUSIONS: These datasets support the existence of a periportal progenitor niche and indicate that clonal patches exhibit punctuated but slow growth, then quiesce, likely due to acute environmental stimuli. These findings crucially contribute to our understanding of hepatocyte dynamics in the normal human liver. IMPACT AND IMPLICATIONS: The liver is mainly composed of hepatocytes, but we know little regarding the source of these cells or how they multiply over time within the disease-free human liver. In this study, we determine a source of new hepatocytes by combining many different lab-based methods and computational predictions to show that hepatocytes share a common cell of origin with bile ducts. Both our experimental and computational data also demonstrate hepatocyte clones are likely to expand in slow waves across the liver in a specific trajectory, but often lie dormant for many years. These data show for the first time the expansion dynamics of hepatocytes in normal liver and their cell of origin enabling the accurate measurment of changes to their dynamics that may lead to liver disease. These findings are important for researchers determining cancer risk in human liver.

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.

Evolutionary history of human colitis-associated colorectal cancer.

OBJECTIVE: IBD confers an increased lifetime risk of developing colorectal cancer (CRC), and colitis-associated CRC (CA-CRC) is molecularly distinct from sporadic CRC (S-CRC). Here we have dissected the evolutionary history of CA-CRC using multiregion sequencing. DESIGN: Exome sequencing was performed on fresh-frozen multiple regions of carcinoma, adjacent non-cancerous mucosa and blood from 12 patients with CA-CRC (n=55 exomes), and key variants were validated with orthogonal methods. Genome-wide copy number profiling was performed using single nucleotide polymorphism arrays and low-pass whole genome sequencing on archival non-dysplastic mucosa (n=9), low-grade dysplasia (LGD; n=30), high-grade dysplasia (HGD; n=13), mixed LGD/HGD (n=7) and CA-CRC (n=19). Phylogenetic trees were reconstructed, and evolutionary analysis used to reveal the temporal sequence of events leading to CA-CRC. RESULTS: 10/12 tumours were microsatellite stable with a median mutation burden of 3.0 single nucleotide alterations (SNA) per Mb, ~20% higher than S-CRC (2.5 SNAs/Mb), and consistent with elevated ageing-associated mutational processes. Non-dysplastic mucosa had considerable mutation burden (median 47 SNAs), including mutations shared with the neighbouring CA-CRC, indicating a precancer mutational field. CA-CRCs were often near triploid (40%) or near tetraploid (20%) and phylogenetic analysis revealed that copy number alterations (CNAs) began to accrue in non-dysplastic bowel, but the LGD/HGD transition often involved a punctuated 'catastrophic' CNA increase. CONCLUSIONS: Evolutionary genomic analysis revealed precancer clones bearing extensive SNAs and CNAs, with progression to cancer involving a dramatic accrual of CNAs at HGD. Detection of the cancerised field is an encouraging prospect for surveillance, but punctuated evolution may limit the window for early detection.

Superficial Esophageal Mucosal Afferent Nerves May Contribute to Reflux Hypersensitivity in Nonerosive Reflux Disease.

BACKGROUND & AIMS: Little is known about the causes of heartburn in patients with gastro-esophageal reflux disease. Visible epithelial damage is seldom associated with symptom severity, evidenced by the significant symptom burden in patients with nonerosive reflux disease (NERD) compared with patients with erosive reflux disease (ERD) or Barrett's esophagus (BE). We studied the distribution of mucosal nerve fibers in patients with NERD, ERD, and BE, and compared the results with those of healthy subjects. METHODS: We performed a prospective study of 13 patients with NERD, 11 patients with ERD, and 16 patients with BE undergoing endoscopic evaluation in the United Kingdom or Greece. Biopsies were obtained from the proximal and distal esophageal mucosa of patients with NERD, from the distal esophageal mucosa of patients with ERD, and the distal-most squamous epithelium of patients with BE. These were examined for the presence and location of nerve fibers that reacted with a labeled antibody against calcitonin gene-related peptide (CGRP), a marker of nociceptive sensory nerves. The results were compared with those from 10 healthy volunteers (controls). RESULTS: The distribution of CGRP-positive nerves did not differ significantly between the distal esophageal mucosa of controls (median, 25.5 cell layers to surface; interquartile range [IQR], 21.4-28.8) vs patients with ERD (median, 23 cell layers to surface; IQR, 16-27.5), or patients with BE (median, 21.5 cell layers to surface; IQR, 16.1-27.5). However, CGRP-positive nerves were significantly more superficial in mucosa from patients with NERD-both distal (median, 9.5 cell layers to surface; IQR, 1.5-13.3; P < .0001 vs ERD, BE, and controls) and proximal (median, 5.0 cell layers to surface; IQR, 2.5-9.3 vs median 10.4 cell layers to surface; IQR, 8.0-16.9; P = .0098 vs controls). CONCLUSIONS: Proximal and distal esophageal mucosa of patients with NERD have more superficial afferent nerves compared with controls or patients with ERD or BE. Acid hypersensitivity in patients with NERD might be partially explained by the increased proximity of their afferent nerves to the esophageal lumen, and therefore greater exposure to noxious substances in refluxate.

Evolution of oesophageal adenocarcinoma from metaplastic columnar epithelium without goblet cells in Barrett's oesophagus.

OBJECTIVE: Barrett's oesophagus commonly presents as a patchwork of columnar metaplasia with and without goblet cells in the distal oesophagus. The presence of metaplastic columnar epithelium with goblet cells on oesophageal biopsy is a marker of cancer progression risk, but it is unclear whether clonal expansion and progression in Barrett's oesophagus is exclusive to columnar epithelium with goblet cells. DESIGN: We developed a novel method to trace the clonal ancestry of an oesophageal adenocarcinoma across an entire Barrett's segment. Clonal expansions in Barrett's mucosa were identified using cytochrome c oxidase enzyme histochemistry. Somatic mutations were identified through mitochondrial DNA sequencing and single gland whole exome sequencing. RESULTS: By tracing the clonal origin of an oesophageal adenocarcinoma across an entire Barrett's segment through a combination of histopathological spatial mapping and clonal ordering, we find that this cancer developed from a premalignant clonal expansion in non-dysplastic ('cardia-type') columnar metaplasia without goblet cells. CONCLUSION: Our data demonstrate the premalignant potential of metaplastic columnar epithelium without goblet cells in the context of Barrett's oesophagus.

The Complex, Clonal, and Controversial Nature of Barrett's Esophagus.

Barrett's esophagus (BO) is a preneoplastic condition described as the replacement of the stratified squamous epithelium of the distal esophagus with one that histologically presents as a diverse mixture of metaplastic glands resembling gastric or intestinal-type columnar epithelium. The clonal origins of BO are still unclear. More recently, we have begun to investigate the relationship between the various metaplastic gland phenotypes observed in BO, how they evolve, and the cancer risk they bestow. Studies have revealed that glands along the BO segment are clonal units containing a single stem cell clone that can give rise to all the differentiated epithelial cell types in glands. Clonal lineage tracing analysis has revealed that Barrett's glands are capable of bifurcation and this facilitates clonal expansion and competition. In fact, BO in some patients appears to consist of multiple, independently initiated clones that compete with each other for space and possibly resources. This chapter discusses the concepts of clonal competition and expansion in BO and sets out to query what we know about the role of gland diversity and phenotypic evolution within this complex columnar metaplasia.

Lineage tracing reveals multipotent stem cells maintain human adenomas and the pattern of clonal expansion in tumor evolution.

The genetic and morphological development of colorectal cancer is a paradigm for tumorigenesis. However, the dynamics of clonal evolution underpinning carcinogenesis remain poorly understood. Here we identify multipotential stem cells within human colorectal adenomas and use methylation patterns of nonexpressed genes to characterize clonal evolution. Numerous individual crypts from six colonic adenomas and a hyperplastic polyp were microdissected and characterized for genetic lesions. Clones deficient in cytochrome c oxidase (CCO(-)) were identified by histochemical staining followed by mtDNA sequencing. Topographical maps of clone locations were constructed using a combination of these data. Multilineage differentiation within clones was demonstrated by immunofluorescence. Methylation patterns of adenomatous crypts were determined by clonal bisulphite sequencing; methylation pattern diversity was compared with a mathematical model to infer to clonal dynamics. Individual adenomatous crypts were clonal for mtDNA mutations and contained both mucin-secreting and neuroendocrine cells, demonstrating that the crypt contained a multipotent stem cell. The intracrypt methylation pattern was consistent with the crypts containing multiple competing stem cells. Adenomas were epigenetically diverse populations, suggesting that they were relatively mitotically old populations. Intratumor clones typically showed less diversity in methylation pattern than the tumor as a whole. Mathematical modeling suggested that recent clonal sweeps encompassing the whole adenoma had not occurred. Adenomatous crypts within human tumors contain actively dividing stem cells. Adenomas appeared to be relatively mitotically old populations, pocketed with occasional newly generated subclones that were the result of recent rapid clonal expansion. Relative stasis and occasional rapid subclone growth may characterize colorectal tumorigenesis.

The stem cell organisation, and the proliferative and gene expression profile of Barrett's epithelium, replicates pyloric-type gastric glands.

OBJECTIVE: Barrett's oesophagus shows appearances described as 'intestinal metaplasia', in structures called 'crypts' but do not typically display crypt architecture. Here, we investigate their relationship to gastric glands. METHODS: Cell proliferation and migration within Barrett's glands was assessed by Ki67 and iododeoxyuridine (IdU) labelling. Expression of mucin core proteins (MUC), trefoil family factor (TFF) peptides and LGR5 mRNA was determined by immunohistochemistry or by in situ hybridisation, and clonality was elucidated using mitochondrial DNA (mtDNA) mutations combined with mucin histochemistry. RESULTS: Proliferation predominantly occurs in the middle of Barrett's glands, diminishing towards the surface and the base: IdU dynamics demonstrate bidirectional migration, similar to gastric glands. Distribution of MUC5AC, TFF1, MUC6 and TFF2 in Barrett's mirrors pyloric glands and is preserved in Barrett's dysplasia. MUC2-positive goblet cells are localised above the neck in Barrett's glands, and TFF3 is concentrated in the same region. LGR5 mRNA is detected in the middle of Barrett's glands suggesting a stem cell niche in this locale, similar to that in the gastric pylorus, and distinct from gastric intestinal metaplasia. Gastric and intestinal cell lineages within Barrett's glands are clonal, indicating derivation from a single stem cell. CONCLUSIONS: Barrett's shows the proliferative and stem cell architecture, and pattern of gene expression of pyloric gastric glands, maintained by stem cells showing gastric and intestinal differentiation: neutral drift may suggest that intestinal differentiation advances with time, a concept critical for the understanding of the origin and development of Barrett's oesophagus.

Cell migration leads to spatially distinct but clonally related airway cancer precursors.

BACKGROUND: Squamous cell carcinoma of the lung is a common cancer with 95% mortality at 5 years. These cancers arise from preinvasive lesions, which have a natural history of development progressing through increasing severity of dysplasia to carcinoma in situ (CIS), and in some cases, ending in transformation to invasive carcinoma. Synchronous preinvasive lesions identified at autopsy have been previously shown to be clonally related. METHODS: Using autofluorescence bronchoscopy that allows visual observation of preinvasive lesions within the upper airways, together with molecular profiling of biopsies using gene sequencing and loss-of-heterozygosity analysis from both preinvasive lesions and from intervening normal tissue, we have monitored individual lesions longitudinally and documented their visual, histological and molecular relationship. RESULTS: We demonstrate that rather than forming a contiguous field of abnormal tissue, clonal CIS lesions can develop at multiple anatomically discrete sites over time. Further, we demonstrate that patients with CIS in the trachea have invariably had previous lesions that have migrated proximally, and in one case, into the other lung over a period of 12 years. CONCLUSIONS: Molecular information from these unique biopsies provides for the first time evidence that field cancerisation of the upper airways can occur through cell migration rather than via local contiguous cellular expansion as previously thought. Our findings urge a clinical strategy of ablating high-grade premalignant airway lesions with subsequent attentive surveillance for recurrence in the bronchial tree.

Identification of lineage-uncommitted, long-lived, label-retaining cells in healthy human esophagus and stomach, and in metaplastic esophagus.

BACKGROUND & AIMS: The existence of slowly cycling, adult stem cells has been challenged by the identification of actively cycling cells. We investigated the existence of uncommitted, slowly cycling cells by tracking 5-iodo-2'-deoxyuridine (IdU) label-retaining cells (LRCs) in normal esophagus, Barrett's esophagus (BE), esophageal dysplasia, adenocarcinoma, and healthy stomach tissues from patients. METHODS: Four patients (3 undergoing esophagectomy, 1 undergoing esophageal endoscopic mucosal resection for dysplasia and an esophagectomy for esophageal adenocarcinoma) received intravenous infusion of IdU (200 mg/m(2) body surface area; maximum dose, 400 mg) over a 30-minute period; the IdU had a circulation half-life of 8 hours. Tissues were collected at 7, 11, 29, and 67 days after infusion, from regions of healthy esophagus, BE, dysplasia, adenocarcinoma, and healthy stomach; they were analyzed by in situ hybridization, flow cytometry, and immunohistochemical analyses. RESULTS: No LRCs were found in dysplasias or adenocarcinomas, but there were significant numbers of LRCs in the base of glands from BE tissue, in the papillae of the basal layer of the esophageal squamous epithelium, and in the neck/isthmus region of healthy stomach. These cells cycled slowly because IdU was retained for at least 67 days and co-labeling with Ki-67 was infrequent. In glands from BE tissues, most cells did not express defensin-5, Muc-2, or chromogranin A, indicating that they were not lineage committed. Some cells labeled for endocrine markers and IdU at 67 days; these cells represented a small population (<0.1%) of epithelial cells at this time point. The epithelial turnover time of the healthy esophageal mucosa was approximately 11 days (twice that of the intestine). CONCLUSIONS: LRCs of human esophagus and stomach have many features of stem cells (long lived, slow cycling, uncommitted, and multipotent), and can be found in a recognized stem cell niche. Further analyses of these cells, in healthy and metaplastic epithelia, is required.

Crypt dysplasia in Barrett's oesophagus shows clonal identity between crypt and surface cells.

Epithelial dysplasia is an important histological diagnosis signifying the presence of pre-invasive disease, usually needing intervention. However, the specific genetic changes responsible for the induction of this phenotypic change are unknown. Moreover, recent reports indicate that the dysplastic phenotype may not be immutable: in basal crypt dysplasia (CD), unequivocal dysplastic changes are seen in the crypts in Barrett's oesophagus and other pre-invasive lesions in the gastrointestinal tract, but the upper crypts and surface epithelium associated with these dysplastic crypts show the definitive morphology of a differentiated epithelium. The genotypic relationship between CD and the differentiated surface epithelium is presently unclear. We obtained 17 examples of CD: the lower and upper crypts and surface epithelium were differentially laser-microdissected from formalin-fixed, paraffin-embedded sections and mutations were sought in tumour suppressor genes frequently associated with progression in Barrett's oesophagus. We found two patients who both showed a c. C238T mutation in the CDKN2A (CDKN2AInk4A) gene and where the precise microanatomical relationships could be discerned: this mutation was present in both the CD at the crypt base and in the upper crypt and surface epithelium. We conclude that, in CD, the dysplastic basal crypt epithelium and the upper crypt and surface epithelium show clonal CDKN2A mutations, thus showing definitively that the surface epithelium is derived from the dysplastic crypt epithelium: the dysplastic phenotype is therefore not fixed and can be reversed. The mechanism of this change is unclear but may be related to the possibility that dysplastic cells can, probably early in their progression, respond to differentiation signals. However, it is also clear that a heavy mutational burden can be borne by crypts in the gastrointestinal tract without the development of phenotypic dysplasia. We are evidently some way from understanding the plasticity and the genotypic correlates of the dysplastic phenotype.

Field cancerization in the intestinal epithelium of patients with Crohn's ileocolitis.

BACKGROUND & AIMS: Tumors that develop in patients with Crohn's disease tend be multifocal, so field cancerization (the replacement of normal cells with nondysplastic but tumorigenic clones) might contribute to intestinal carcinogenesis. We investigated patterns of tumor development from pretumor intestinal cell clones. METHODS: We performed genetic analyses of multiple areas of intestine from 10 patients with Crohn's disease and intestinal neoplasia. Two patients had multifocal neoplasia; longitudinal sections were collected from 3 patients. Individual crypts were microdissected and genotyped; clonal dependency analysis was used to determine the order and timing of mutations that led to tumor development. RESULTS: The same mutations in KRAS, CDKN2A(p16), and TP53 that were observed in neoplasias were also present in nontumor, nondysplastic, and dysplastic epithelium. In 2 patients, carcinogenic mutations were detected in nontumor epithelium 4 years before tumors developed. The same mutation (TP53 p.R248W) was detected at multiple sites along the entire length of the colon from 1 patient; it was the apparent founder mutation for synchronous tumors and multiple dysplastic areas. Disruption of TP53, CDKN2A, and KRAS were all seen as possible initial events in tumorigenesis; the sequence of mutations (the tumor development pathway) differed among lesions. CONCLUSIONS: Pretumor clones can grow extensively in the intestinal epithelium of patients with Crohn's disease. Segmental resections for neoplasia in patients with Crohn's disease might therefore leave residual pretumor disease, and dysplasia might be an unreliable biomarker for cancer risk. Characterization of the behavior of pretumor clones might be used to predict the development of intestinal neoplasia.

Clonal selection and persistence in dysplastic Barrett's esophagus and intramucosal cancers after failed radiofrequency ablation.

OBJECTIVES: Radiofrequency ablation (RFA) is used to successfully eliminate Barrett's esophagus (BE)-related dysplasia or intramucosal carcinoma and aims to cause reversion to squamous epithelium. However, in 20% of cases RFA fails to return the epithelium to squamous phenotype. Follow-up studies show a similar dysplasia recurrence rate. We hypothesize that failed RFA is due to clonally mutated epithelial populations harbored in RFA-privileged sites and that RFA can select for the mutant clonal expansion. METHODS: A longitudinal case series of 19 patients with BE and high-grade dysplasia or intramucosal carcinoma were studied. DNA was extracted from individual Barrett's glands, deep esophageal glands within mucosal resections and biopsy specimens before and after RFA. Mutations were identified by targeted sequencing of genes commonly mutated in Barrett's adenocarcinoma. RESULTS: Five patients demonstrated persistent post-RFA pathology with persistent mutations, sometimes detected in deep esophageal glands or neighboring squamous epithelium after several rounds of RFA preceded by mucosal resection. Recurrence of pathology in three other patients was characterized by de novo mutations. CONCLUSIONS: Protumorigenic mutations can be found in post-ablation squamous mucosa as well as in mutant deep esophageal glands; both are associated with dysplasia recurrence. Following RFA, non-dysplastic Barrett's epithelium can contain mutant clones that are found in a subsequent adenocarcinoma. Ablation may also drive the clonal expansion of pre-existing clones after a "bottleneck" created by the RFA. Overall, recurrence of dysplasia post RFA reflects the multicentric origins of Barrett's clones and highlights the role of clonal selection in carcinogenesis.

Barrett's metaplasia glands are clonal, contain multiple stem cells and share a common squamous progenitor.

BACKGROUND: Little is known about the stem cell organisation of the normal oesophagus or Barrett's metaplastic oesophagus. Using non-pathogenic mitochondrial DNA mutations as clonal markers, the authors reveal the stem cell organisation of the human squamous oesophagus and of Barrett's metaplasia and determine the mechanism of clonal expansion of mutations. METHODS: Mutated cells were identified using enzyme histochemistry to detect activity of cytochrome c oxidase (CCO). CCO-deficient cells were laser-captured and mutations confirmed by PCR sequencing. Cell lineages were identified using immunohistochemistry. RESULTS: The normal squamous oesophagus contained CCO-deficient patches varying in size from around 30 µm up to about 1 mm. These patches were clonal as each area within a CCO-deficient patch contained an identical mitochondrial DNA mutation. In Barrett's metaplasia partially CCO-deficient glands indicate that glands are maintained by multiple stem cells. Wholly mutated Barrett's metaplasia glands containing all the expected differentiated cell lineages were seen, demonstrating multilineage differentiation from a clonal population of Barrett's metaplasia stem cells. Patches of clonally mutated Barrett's metaplasia glands were observed, indicating glands can divide to form patches. In one patient, both the regenerating squamous epithelium and the underlying glandular tissue shared a clonal mutation, indicating that they are derived from a common progenitor cell. CONCLUSION: In normal oesophageal squamous epithelium, a single stem cell clone can populate large areas of epithelium. Barrett's metaplasia glands are clonal units, contain multiple multipotential stem cells and most likely divide by fission. Furthermore, a single cell of origin can give rise to both squamous and glandular epithelium suggesting oesophageal plasticity.

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.

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.

The human urothelium consists of multiple clonal units, each maintained by a stem cell.

Little is known about the clonal architecture of human urothelium. It is likely that urothelial stem cells reside within the basal epithelial layer, yet lineage tracing from a single stem cell as a means to show the presence of a urothelial stem cell has never been performed. Here, we identify clonally related cell areas within human bladder mucosa in order to visualize epithelial fields maintained by a single founder/stem cell. Sixteen frozen cystectomy specimens were serially sectioned. Patches of cells deficient for the mitochondrially encoded enzyme cytochrome c oxidase (CCO) were identified using dual-colour enzyme histochemistry. To show that these patches represent clonal proliferations, small CCO-proficient and -deficient areas were individually laser-capture microdissected and the entire mitochondrial genome (mtDNA) in each area was PCR amplified and sequenced to identify mtDNA mutations. Immunohistochemistry was performed for the different cell layers of the urothelium and adjacent mesenchyme. CCO-deficient patches could be observed in normal urothelium of all cystectomy specimens. The two-dimensional length of these negative patches varied from 2-3 cells (about 30 µm) to 4.7 mm. Each cell area within a CCO-deficient patch contained an identical somatic mtDNA mutation, indicating that the patch was a clonal unit. Patches contained all the mature cell differentiation stages present in the urothelium, suggesting the presence of a stem cell. Our results demonstrate that the normal mucosa of human bladder contains stem cell-derived clonal units that actively replenish the urothelium during ageing. The size of the clonal unit attributable to each stem cell was broadly distributed, suggesting replacement of one stem cell clone by another.

Evolutionary dynamics in Barrett oesophagus: implications for surveillance, risk stratification and therapy.

Cancer development is a dynamic evolutionary process characterized by marked intratumoural heterogeneity at the genetic, epigenetic and phenotypic levels. Barrett oesophagus, the pre-malignant condition to oesophageal adenocarcinoma (EAC), is an exemplary system to longitudinally study the evolution of malignancy. Evidence has emerged of Barrett oesophagus lesions pre-programmed for progression to EAC many years before clinical detection, indicating a considerable window for therapeutic intervention. In this Review, we explore the mechanisms underlying clonal expansion and contraction that establish the Barrett oesophagus clonal mosaicism over time and space and discuss intrinsic genotypic and extrinsic environmental drivers that direct the evolutionary trajectory of Barrett oesophagus towards a malignant phenotype. We propose that understanding and exploiting the evolutionary dynamics of Barrett oesophagus will identify novel therapeutic targets, improve prognostic tools and offer the opportunity for personalized surveillance programmes geared to prevent progression to EAC.

Cell Competition in Carcinogenesis.

The majority of human cancers evolve over time through the stepwise accumulation of somatic mutations followed by clonal selection akin to Darwinian evolution. However, the in-depth mechanisms that govern clonal dynamics and selection remain elusive, particularly during the earliest stages of tissue transformation. Cell competition (CC), often referred to as 'survival of the fittest' at the cellular level, results in the elimination of less fit cells by their more fit neighbors supporting optimal organism health and function. Alternatively, CC may allow an uncontrolled expansion of super-fit cancer cells to outcompete their less fit neighbors thereby fueling tumorigenesis. Recent research discussed herein highlights the various non-cell-autonomous principles, including interclonal competition and cancer microenvironment competition supporting the ability of a tumor to progress from the initial stages to tissue colonization. In addition, we extend current insights from CC-mediated clonal interactions and selection in normal tissues to better comprehend those factors that contribute to cancer development.

Clonality assessment and clonal ordering of individual neoplastic crypts shows polyclonality of colorectal adenomas.

BACKGROUND & AIMS: According to the somatic mutation theory, monoclonal colorectal lesions arise from sequential mutations in the progeny of a single stem cell. However, studies in a sex chromosome mixoploid mosaic (XO/XY) patient indicated that colorectal adenomas were polyclonal. We assessed adenoma clonality on an individual crypt basis and completed a genetic dependency analysis in carcinomas-in-adenomas to assess mutation order and timing. METHODS: Polyp samples were analyzed from the XO/XY individual, patients with familial adenomatous polyposis and attenuated familial adenomatous polyposis, patients with small sporadic adenomas, and patients with sporadic carcinoma-in-adenomas. Clonality was analyzed using X/Y chromosome fluorescence in situ hybridization, analysis of 5q loss of heterozygosity in XO/XY tissue, and sequencing of adenomatous polyposis coli. Individual crypts and different phenotypic areas of carcinoma-in-adenoma lesions were analyzed for mutations in adenomatous polyposis coli, p53, and K-RAS; loss of heterozygosity at 5q, 17p, and 18q; and aneuploidy. Phylogenetic trees were constructed. RESULTS: All familial adenomatous polyposis-associated adenomas and some sporadic lesions had polyclonal genetic defects. Some independent clones appeared to be maintained in advanced adenomas. No clear obligate order of genetic events was established. Top-down growth of dysplastic tissue into neighboring crypts was a possible mechanism of clonal competition. CONCLUSIONS: Human colorectal microadenomas are polyclonal and may arise from a combination of host genetic features, mucosal exposures, and active crypt interactions. Analyses of tumor phylogenies show that most lesions undergo intermittent genetic homogenization, but heterotypic mutation patterns indicate that independent clonal evolution can occur throughout adenoma development. Based on observations of clonal ordering the requirement and timing of genetic events during neoplastic progression may be more variable than previously thought.