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.

Lineage tracing in human tissues.

The dynamical process of cell division that underpins homeostasis in the human body cannot be directly observed in vivo, but instead is measurable from the pattern of somatic genetic or epigenetic mutations that accrue in tissues over an individual's lifetime. Because somatic mutations are heritable, they serve as natural lineage tracing markers that delineate clonal expansions. Mathematical analysis of the distribution of somatic clone sizes gives a quantitative readout of the rates of cell birth, death, and replacement. In this review we explore the broad range of somatic mutation types that have been used for lineage tracing in human tissues, introduce the mathematical concepts used to infer dynamical information from these clone size data, and discuss the insights of this lineage tracing approach for our understanding of homeostasis and cancer development. We use the human colon as a particularly instructive exemplar tissue. There is a rich history of human somatic cell dynamics surreptitiously written into the cell genomes that is being uncovered by advances in sequencing and careful mathematical analysis lineage of tracing data. © 2022 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

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.

Lrig1 expression identifies airway basal cells with high proliferative capacity and restricts lung squamous cell carcinoma growth.

BACKGROUND: Lung squamous cell carcinoma (LUSC) accounts for a significant proportion of cancer deaths worldwide, and is preceded by the appearance of progressively disorganised pre-invasive lesions in the airway epithelium. Yet the biological mechanisms underlying progression of pre-invasive lesions into invasive LUSC are not fully understood. LRIG1 (leucine-rich repeats and immunoglobulin-like domains 1) is downregulated in pre-invasive airway lesions and invasive LUSC tumours and this correlates with decreased lung cancer patient survival. METHODS AND RESULTS: Using an Lrig1 knock-in reporter mouse and human airway epithelial cells collected at bronchoscopy, we show that during homeostasis LRIG1 is heterogeneously expressed in the airway epithelium. In basal airway epithelial cells, the suspected cell of origin of LUSC, LRIG1 identifies a subpopulation of progenitor cells with higher in vitro proliferative and self-renewal potential in both the mouse and human. Using the N-nitroso-tris-chloroethylurea (NTCU)-induced murine model of LUSC, we find that Lrig1 loss-of-function leads to abnormally high cell proliferation during the earliest stages of pre-invasive disease and to the formation of significantly larger invasive tumours, suggesting accelerated disease progression. CONCLUSION: Together, our findings identify LRIG1 as a marker of basal airway progenitor cells with high proliferative potential and as a regulator of pre-invasive lung cancer progression. This work highlights the clinical relevance of LRIG1 and the potential of the NTCU-induced LUSC model for functional assessment of candidate tumour suppressors and oncogenes.

Histological 3D reconstruction and in vivo lineage tracing of the human endometrium.

Regular menstrual shedding and repair of the endometrial functionalis is unique to humans and higher-order primates. The current consensus postulates endometrial glands to have a single-tubular architecture, where multi-potential stem cells reside in the blind-ending glandular-bases. Utilising fixed samples from patients, we have studied the three-dimensional (3D) micro-architecture of the human endometrium. We demonstrate that some non-branching, single, vertical functionalis glands originate from a complex horizontally interconnecting network of basalis glands. The existence of a multipotent endometrial epithelial stem cell capable of regenerating the entire complement of glandular lineages was demonstrated by in vivo lineage tracing, using naturally occurring somatic mitochondrial DNA mutations as clonal markers. Vertical tracking of mutated clones showed that at least one stem-cell population resides in the basalis glands. These novel findings provide insight into the efficient and scar-less regenerative potential of the human endometrium. © 2020 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

Crypt fusion as a homeostatic mechanism in the human colon.

OBJECTIVE: The crypt population in the human intestine is dynamic: crypts can divide to produce two new daughter crypts through a process termed crypt fission, but whether this is balanced by a second process to remove crypts, as recently shown in mouse models, is uncertain. We examined whether crypt fusion (the process of two neighbouring crypts fusing into a single daughter crypt) occurs in the human colon. DESIGN: We used somatic alterations in the gene cytochrome c oxidase (CCO) as lineage tracing markers to assess the clonality of bifurcating colon crypts (n=309 bifurcating crypts from 13 patients). Mathematical modelling was used to determine whether the existence of crypt fusion can explain the experimental data, and how the process of fusion influences the rate of crypt fission. RESULTS: In 55% (21/38) of bifurcating crypts in which clonality could be assessed, we observed perfect segregation of clonal lineages to the respective crypt arms. Mathematical modelling showed that this frequency of perfect segregation could not be explained by fission alone (p<10-20). With the rates of fission and fusion taken to be approximately equal, we then used the distribution of CCO-deficient patch size to estimate the rate of crypt fission, finding a value of around 0.011 divisions/crypt/year. CONCLUSIONS: We have provided the evidence that human colonic crypts undergo fusion, a potential homeostatic process to regulate total crypt number. The existence of crypt fusion in the human colon adds a new facet to our understanding of the highly dynamic and plastic phenotype of the colonic epithelium.

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.

Is Barrett's-Associated Esophageal Adenocarcinoma a Clonal Disease?

In this study, we argue that the basic clonal unit that makes up the Barrett's segment is at the level of the gland. There is expansion of this clonal unit, the gland, by fission, and there is evidence that the Barrett's segment is itself a clonal proliferation. Barrett's esophagus arises from both goblet cell-containing metaplasia and non-goblet cell-containing metaplasia and may arise from a stable clone, but the genomic changes occurring are subject to selection, usually with little or no evolution, appearing indolent from the evolutionary perspective. Genomic changes leading to dysplastic phenotypes are selected, but without any single clone predominating within the segment.

Analysis of clonal expansions through the normal and premalignant human breast epithelium reveals the presence of luminal stem cells.

It is widely accepted that the cell of origin of breast cancer is the adult mammary epithelial stem cell; however, demonstrating the presence and location of tissue stem cells in the human breast has proved difficult. Furthermore, we do not know the clonal architecture of the normal and premalignant mammary epithelium or its cellular hierarchy. Here, we use deficiency in the mitochondrial enzyme cytochrome c oxidase (CCO), typically caused by somatic mutations in the mitochondrial genome, as a means to perform lineage tracing in the human mammary epithelium. PCR sequencing of laser-capture microdissected cells in combination with immunohistochemistry for markers of lineage differentiation was performed to determine the clonal nature of the mammary epithelium. We have shown that in the normal human breast, clonal expansions (defined here by areas of CCO deficiency) are typically uncommon and of limited size, but can occur at any site within the adult mammary epithelium. The presence of a stem cell population was shown by demonstrating multi-lineage differentiation within CCO-deficient areas. Interestingly, we observed infrequent CCO deficiency that was restricted to luminal cells, suggesting that niche succession, and by inference stem cell location, is located within the luminal layer. CCO-deficient areas appeared large within areas of ductal carcinoma in situ, suggesting that the rate of clonal expansion was altered in the premalignant lesion. © 2017 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

An evolutionary perspective on field cancerization.

Tumorigenesis begins long before the growth of a clinically detectable lesion and, indeed, even before any of the usual morphological correlates of pre-malignancy are recognizable. Field cancerization, which is the replacement of the normal cell population by a cancer-primed cell population that may show no morphological change, is now recognized to underlie the development of many types of cancer, including the common carcinomas of the lung, colon, skin, prostate and bladder. Field cancerization is the consequence of the evolution of somatic cells in the body that results in cells that carry some but not all phenotypes required for malignancy. Here, we review the evidence of field cancerization across organs and examine the biological mechanisms that drive the evolutionary process that results in field creation. We discuss the clinical implications, principally, how measurements of the cancerized field could improve cancer risk prediction in patients with pre-malignant disease.

Robust RNA-based in situ mutation detection delineates colorectal cancer subclonal evolution.

Intra-tumor heterogeneity (ITH) is a major underlying cause of therapy resistance and disease recurrence, and is a read-out of tumor growth. Current genetic ITH analysis methods do not preserve spatial context and may not detect rare subclones. Here, we address these shortfalls by developing and validating BaseScope-a novel mutation-specific RNA in situ hybridization assay. We target common point mutations in the BRAF, KRAS and PIK3CA oncogenes in archival colorectal cancer samples to precisely map the spatial and morphological context of mutant subclones. Computational modeling suggests that subclones must arise sufficiently early, or carry a considerable fitness advantage, to form large or spatially disparate subclones. Examples of putative treatment-resistant cells isolated in small topographical areas are observed. The BaseScope assay represents a significant technical advance for in situ mutation detection that provides new insight into tumor evolution, and could have ramifications for selecting patients for treatment.

Gastrin Induces Nuclear Export and Proteasome Degradation of Menin in Enteric Glial Cells.

BACKGROUND & AIMS: The multiple endocrine neoplasia, type 1 (MEN1) locus encodes the nuclear protein and tumor suppressor menin. MEN1 mutations frequently cause neuroendocrine tumors such as gastrinomas, characterized by their predominant duodenal location and local metastasis at time of diagnosis. Diffuse gastrin cell hyperplasia precedes the appearance of MEN1 gastrinomas, which develop within submucosal Brunner's glands. We investigated how menin regulates expression of the gastrin gene and induces generation of submucosal gastrin-expressing cell hyperplasia. METHODS: Primary enteric glial cultures were generated from the VillinCre:Men1FL/FL:Sst-/- mice or C57BL/6 mice (controls), with or without inhibition of gastric acid by omeprazole. Primary enteric glial cells from C57BL/6 mice were incubated with gastrin and separated into nuclear and cytoplasmic fractions. Cells were incubated with forskolin and H89 to activate or inhibit protein kinase A (a family of enzymes whose activity depends on cellular levels of cyclic AMP). Gastrin was measured in blood, tissue, and cell cultures using an ELISA. Immunoprecipitation with menin or ubiquitin was used to demonstrate post-translational modification of menin. Primary glial cells were incubated with leptomycin b and MG132 to block nuclear export and proteasome activity, respectively. We obtained human duodenal, lymph node, and pancreatic gastrinoma samples, collected from patients who underwent surgery from 1996 through 2007 in the United States or the United Kingdom. RESULTS: Enteric glial cells that stained positive for glial fibrillary acidic protein (GFAP+) expressed gastrin de novo through a mechanism that required PKA. Gastrin-induced nuclear export of menin via cholecystokinin B receptor (CCKBR)-mediated activation of PKA. Once exported from the nucleus, menin was ubiquitinated and degraded by the proteasome. GFAP and other markers of enteric glial cells (eg, p75 and S100B), colocalized with gastrin in human duodenal gastrinomas. CONCLUSIONS: MEN1-associated gastrinomas, which develop in the submucosa, might arise from enteric glial cells through hormone-dependent PKA signaling. This pathway disrupts nuclear menin function, leading to hypergastrinemia and associated sequelae.

Multipotent Basal Stem Cells, Maintained in Localized Proximal Niches, Support Directed Long-Ranging Epithelial Flows in Human Prostates.

Sporadic mitochondrial DNA mutations serve as clonal marks providing access to the identity and lineage potential of stem cells within human tissues. By combining quantitative clonal mapping with 3D reconstruction of adult human prostates, we show that multipotent basal stem cells, confined to discrete niches in juxta-urethral ducts, generate bipotent basal progenitors in directed epithelial migration streams. Basal progenitors are then dispersed throughout the entire glandular network, dividing and differentiating to replenish the loss of apoptotic luminal cells. Rare lineage-restricted luminal stem cells, and their progeny, are confined to proximal ducts and provide only minor contribution to epithelial homeostasis. In situ cell capture from clonal maps identified delta homolog 1 (DLK1) enrichment of basal stem cells, which was validated in functional spheroid assays. This study establishes significant insights into niche organization and function of prostate stem and progenitor cells, with implications for disease.

Evolution of Premalignant Disease.

Where does cancer come from? Although the cell-of-origin is difficult to pinpoint, cancer clones harbor information about their clonal ancestries. In an effort to find cells before they evolve into a life-threatening cancer, physicians currently diagnose premalignant diseases at frequencies that substantially exceed those of clinical cancers. Cancer risk prediction relies on our ability to distinguish between which premalignant features will lead to cancer mortality and which are characteristic of inconsequential disease. Here, we review the evolution of cancer from premalignant disease, and discuss the concept that even phenotypically normal cell progenies inherently gain more malignant potential with age. We describe the hurdles of prognosticating cancer risk in premalignant disease by making reference to the underlying continuous and multivariate natures of genotypes and phenotypes and the particular challenge inherent in defining a cell lineage as "cancerized."

Distal Esophageal Adenocarcinoma and Gastric Adenocarcinoma: Time for a Shared Research Agenda.

The key insight that sparked Darwin's theory of descent with modification was that he compared and contrasted differences between living and extinct species across time and space. He likely arrived on this theory in large part through his culinary experiences, set against the background of the rugged Patagonian landscape of Southern Argentina. We feel that further integration of research into gastric and esophageal adenocarcinoma may benefit both fields and similarly lead to a coherent understanding of cancer progression in the upper gastrointestinal tract across time and space. Although the environmental trigger differs between carcinogenesis of the stomach and distal esophagus, there remain many important lessons to be learned from comparing precursor stages, such as intestinal metaplasia, across anatomic borders. This analysis will absolutely require detailed sampling within and between these related species, but most importantly we need higher resolution clinical phenotyping to relate genomic differences to drivers of morphologic evolution. In the end, this may provide us with a new phylogeny showing key differences between esophageal and gastric adenocarcinoma.

New paradigms in clonal evolution: punctuated equilibrium in cancer.

Evolutionary theories are themselves subject to evolution. Clonal evolution - the model that describes the initiation and progression of cancer - is entering a period of profound change, brought about largely by technological developments in genome analysis. A flurry of recent publications, using modern mathematical and bioinformatics techniques, have revealed both punctuated and neutral evolution phenomena that are poorly explained by the conventional graduated perspectives. In this review, we propose that a hybrid model, inspired by the evolutionary model of punctuated equilibrium, could better explain these recent observations. We also discuss the conceptual changes and clinical implications of variable evolutionary tempos. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Distribution of the c-MYC gene product in colorectal neoplasia.

AIMS: Recent attempts to study MYC distribution in human samples have been confounded by a lack of agreement in immunohistochemical staining between antibodies targeting the N-terminus and those targeting the C-terminus of the MYC protein. The aim of this study was to use a novel in-situ hybridization (ISH) approach to detect MYC mRNA in clinically relevant samples, and thereby determine the reliability of MYC-targeting antibodies. METHODS AND RESULTS: We performed immunohistochemistry on human formalin-fixed paraffin embedded normal colon (n = 15), hyperplastic polyp (n = 4) and neoplastic colon samples (n = 55), using the N-terminally directed antibody Y69, and the C-terminally directed antibody 9E10. The MYC protein distributions were then compared with the location of MYC mRNA, determined by ISH. We found that the localization of MYC mRNA correlated well with the protein distribution determined with the N-terminally directed antibody Y69, and was also associated with expression of the proliferation marker Ki67. The protein distribution determined with the C-terminally directed antibody 9E10 was not always associated with MYC mRNA, Y69, or Ki67, and indeed often showed a reciprocal pattern of expression, with staining being strongest in non-proliferating cells. CONCLUSIONS: The observed discrepancy between the staining patterns suggests that the significance of 9E10 in immunohistochemical staining is currently uncertain, and therefore should be interpreted with caution.

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.