The Bowel Cancer Screening Programme Expert Board: an analysis of activity during 2017-2020.

AIMS: The inception of the National Health Service Bowel Cancer Screening Programme in England in 2006 highlighted the fact that the differential diagnosis between the presence of epithelial misplacement and adenocarcinoma occurring in colorectal adenomas is problematic. The pathology Expert Board (EB) was created to facilitate the review of difficult cases by a panel of three experienced gastrointestinal pathologists. This article describes a review of the work of the EB over a 4-year period (2017-2020). METHODS AND RESULTS: Four hundred and thirty polyps were referred to the EB from 193 pathologists and 76 hospitals during this time. The EB diagnosis was benign for 67%, malignant for 28%, and equivocal for 2% (with no consensus in the remainder). The most common diagnosis change made by the EB was from malignant to benign-made in 50% of polyps referred with an initially malignant diagnosis. The level of agreement between the individual EB members was 'good' (kappa score of 0.619) but that between the EB and the referring diagnosis was 'poor' (kappa score of 0.149). Data from one EB member indicated that the presence of lamina propria, features of torsion and cytological similarity between the superficial and deep glands were predictors of a benign diagnosis, whereas the presence of irregular neoplastic glands, a desmoplastic reaction and lymphovascular invasion were commonly observed features in polyps with a malignant diagnosis. CONCLUSION: Diagnostic agreement between EB members is better than that between the EB and referring pathologists. There was a consistent trend for the EB to change diagnoses from malignant to benign.

Current dilemmas in the pathological staging of colorectal cancer: the results of a national survey.

AIMS: Accurate and consistent pathological staging of colorectal carcinoma (CRC) in resection specimens is especially crucial to guide adjuvant therapy. The aim of this study was to assess whether certain staging scenarios yield discordant opinions in the setting of current international and UK national guidelines. METHODS AND RESULTS: Members of the UK Gastrointestinal Pathology External Quality Assurance Scheme were invited to complete an anonymous, on-line survey that presented 15 scenarios related to pT or pR staging of CRC, and three questions about the respondent. The survey invitation was e-mailed to 405 pathologists, and 184 (45%) responses were received. The respondents had discordant opinions on whether and how CRC pT or pR staging is affected by: acellular mucin lakes and duration after short-course radiotherapy; the nature of the carcinoma at a resection margin or peritoneal surface; and microscopic evidence of perforation. This discordance was rarely related to the respondent's occupation type, and was not related to duration of work as a consultant or the staging guidelines used. CONCLUSIONS: This survey confirms that there remain several clinically critical but unresolved pT and pR staging issues for CRC. These issues therefore deserve attention in future versions of international and national staging guidelines.

British Society of Gastroenterology/Association of Coloproctology of Great Britain and Ireland/Public Health England post-polypectomy and post-colorectal cancer resection surveillance guidelines.

These consensus guidelines were jointly commissioned by the British Society of Gastroenterology (BSG), the Association of Coloproctology of Great Britain and Ireland (ACPGBI) and Public Health England (PHE). They provide an evidence-based framework for the use of surveillance colonoscopy and non-colonoscopic colorectal imaging in people aged 18 years and over. They are the first guidelines that take into account the introduction of national bowel cancer screening. For the first time, they also incorporate surveillance of patients following resection of either adenomatous or serrated polyps and also post-colorectal cancer resection. They are primarily aimed at healthcare professionals, and aim to address:Which patients should commence surveillance post-polypectomy and post-cancer resection?What is the appropriate surveillance interval?When can surveillance be stopped? two or more premalignant polyps including at least one advanced colorectal polyp (defined as a serrated polyp of at least 10 mm in size or containing any grade of dysplasia, or an adenoma of at least 10 mm in size or containing high-grade dysplasia); or five or more premalignant polyps The Appraisal of Guidelines for Research and Evaluation (AGREE II) instrument provided a methodological framework for the guidelines. The BSG's guideline development process was used, which is National Institute for Health and Care Excellence (NICE) compliant.two or more premalignant polyps including at least one advanced colorectal polyp (defined as a serrated polyp of at least 10 mm in size or containing any grade of dysplasia, or an adenoma of at least 10 mm in size or containing high-grade dysplasia); or five or more premalignant polyps The key recommendations are that the high-risk criteria for future colorectal cancer (CRC) following polypectomy comprise either:two or more premalignant polyps including at least one advanced colorectal polyp (defined as a serrated polyp of at least 10 mm in size or containing any grade of dysplasia, or an adenoma of at least 10 mm in size or containing high-grade dysplasia); or five or more premalignant polyps This cohort should undergo a one-off surveillance colonoscopy at 3 years. Post-CRC resection patients should undergo a 1 year clearance colonoscopy, then a surveillance colonoscopy after 3 more years.

British Society of Gastroenterology guidelines on the diagnosis and management of patients at risk of gastric adenocarcinoma.

Gastric adenocarcinoma carries a poor prognosis, in part due to the late stage of diagnosis. Risk factors include Helicobacter pylori infection, family history of gastric cancer-in particular, hereditary diffuse gastric cancer and pernicious anaemia. The stages in the progression to cancer include chronic gastritis, gastric atrophy (GA), gastric intestinal metaplasia (GIM) and dysplasia. The key to early detection of cancer and improved survival is to non-invasively identify those at risk before endoscopy. However, although biomarkers may help in the detection of patients with chronic atrophic gastritis, there is insufficient evidence to support their use for population screening. High-quality endoscopy with full mucosal visualisation is an important part of improving early detection. Image-enhanced endoscopy combined with biopsy sampling for histopathology is the best approach to detect and accurately risk-stratify GA and GIM. Biopsies following the Sydney protocol from the antrum, incisura, lesser and greater curvature allow both diagnostic confirmation and risk stratification for progression to cancer. Ideally biopsies should be directed to areas of GA or GIM visualised by high-quality endoscopy. There is insufficient evidence to support screening in a low-risk population (undergoing routine diagnostic oesophagogastroduodenoscopy) such as the UK, but endoscopic surveillance every 3 years should be offered to patients with extensive GA or GIM. Endoscopic mucosal resection or endoscopic submucosal dissection of visible gastric dysplasia and early cancer has been shown to be efficacious with a high success rate and low rate of recurrence, providing that specific quality criteria are met.

Critical research gaps and recommendations to inform research prioritisation for more effective prevention and improved outcomes in colorectal cancer.

OBJECTIVE: Colorectal cancer (CRC) leads to significant morbidity/mortality worldwide. Defining critical research gaps (RG), their prioritisation and resolution, could improve patient outcomes. DESIGN: RG analysis was conducted by a multidisciplinary panel of patients, clinicians and researchers (n=71). Eight working groups (WG) were constituted: discovery science; risk; prevention; early diagnosis and screening; pathology; curative treatment; stage IV disease; and living with and beyond CRC. A series of discussions led to development of draft papers by each WG, which were evaluated by a 20-strong patient panel. A final list of RGs and research recommendations (RR) was endorsed by all participants. RESULTS: Fifteen critical RGs are summarised below: RG1: Lack of realistic models that recapitulate tumour/tumour micro/macroenvironment; RG2: Insufficient evidence on precise contributions of genetic/environmental/lifestyle factors to CRC risk; RG3: Pressing need for prevention trials; RG4: Lack of integration of different prevention approaches; RG5: Lack of optimal strategies for CRC screening; RG6: Lack of effective triage systems for invasive investigations; RG7: Imprecise pathological assessment of CRC; RG8: Lack of qualified personnel in genomics, data sciences and digital pathology; RG9: Inadequate assessment/communication of risk, benefit and uncertainty of treatment choices; RG10: Need for novel technologies/interventions to improve curative outcomes; RG11: Lack of approaches that recognise molecular interplay between metastasising tumours and their microenvironment; RG12: Lack of reliable biomarkers to guide stage IV treatment; RG13: Need to increase understanding of health related quality of life (HRQOL) and promote residual symptom resolution; RG14: Lack of coordination of CRC research/funding; RG15: Lack of effective communication between relevant stakeholders. CONCLUSION: Prioritising research activity and funding could have a significant impact on reducing CRC disease burden over the next 5 years.

Upregulation of mucin glycoprotein MUC1 in the progression to esophageal adenocarcinoma and therapeutic potential with a targeted photoactive antibody-drug conjugate.

BACKGROUND: Mucin glycoprotein 1 (MUC1) is a glycosylated transmembrane protein on epithelial cells. We investigate MUC1 as a therapeutic target in Barrett's epithelium (BE) and esophageal adenocarcinoma (EA) and provide proof of concept for a light based therapy targeting MUC1. RESULTS: MUC1 was present in 21% and 30% of significantly enriched pathways comparing BE and EA to squamous epithelium respectively. MUC1 gene expression was x2.3 and x2.2 higher in BE (p=<0.001) and EA (p=0.03). MUC1 immunohistochemical expression increased during progression to EA and followed tumor invasion. HuHMFG1 based photosensitive antibody drug conjugates (ADC) showed cell internalization, MUC1 selective and light-dependent cytotoxicity (p=0.0006) and superior toxicity over photosensitizer alone (p=0.0022). METHODS: Gene set enrichment analysis (GSEA) evaluated pathways during BE and EA development and quantified MUC1 gene expression. Immunohistochemistry and flow cytometry evaluated the anti-MUC1 antibody HuHMFG1 in esophageal cells of varying pathological grade. Confocal microscopy examined HuHMFG1 internalization and HuHMFG1 ADCs were created to deliver a MUC1 targeted phototoxic payload. CONCLUSIONS: MUC1 is a promising target in EA. Molecular and light based targeting of MUC1 with a photosensitive ADC is effective in vitro and after development may enable treatment of locoregional tumors endoscopically.

Challenging diagnostic issues in adenomatous polyps with epithelial misplacement in bowel cancer screening: 5 years' experience of the Bowel Cancer Screening Programme Expert Board.

The diagnostic difficulties of differentiating epithelial misplacement from invasive cancer in colorectal adenomatous polyps have been recognised for many years. Nevertheless, the introduction of population screening in the UK has resulted in extraordinary diagnostic problems. Larger sigmoid colonic adenomatous polyps, which are those most likely to show epithelial misplacement, are specifically selected into such screening programmes, because these polyps are likely to bleed and screening is based on the detection of occult blood. The diagnostic challenges associated with this particular phenomenon have necessitated the institution of an 'Expert Board': this is a review of the first five years of its practice, during which time 256 polyps from 249 patients have been assessed. Indeed, the Expert Board contains three pathologists, because those pathologists do not necessarily agree, and a consensus diagnosis is required to drive appropriate patient management. However, this study has shown substantial levels of agreement between the three Expert Board pathologists, whereby the ultimate diagnosis has been changed, from that of the original referral diagnosis, by the Expert Board for half of all the polyps, in the substantial majority from malignant to benign. In 3% of polyp cases, the Expert Board consensus has been the dual diagnosis of both epithelial misplacement and adenocarcinoma, further illustrating the diagnostic difficulties. The Expert Board of the Bowel Cancer Screening Programme in the UK represents a unique and successful development in response to an extraordinary diagnostic conundrum created by the particular characteristics of bowel cancer screening.

Diagnosis of T1 colorectal cancer in pedunculated polyps in daily clinical practice: a multicenter study.

T1 colorectal cancer can be mimicked by pseudo-invasion in pedunculated polyps. British guidelines are currently one of the few which recommend diagnostic confirmation of T1 colorectal cancer by a second pathologist. The aim of this study was to provide insights into the accuracy of histological diagnosis of pedunculated T1 colorectal cancer in daily clinical practice. A sample of 128 cases diagnosed as pedunculated T1 colorectal cancer between 2000 and 2014 from 10 Dutch hospitals was selected for histological review. Firstly, two Dutch expert gastrointestinal pathologists reviewed all hematoxylin-eosin stained slides. In 20 cases the diagnosis T1 colorectal cancer was not confirmed (20/128; 16%). The discordant cases were subsequently discussed with a third Dutch gastrointestinal pathologist and a consensus diagnosis was agreed. The revised diagnoses were pseudo-invasion in 10 cases (10/128; 8%), high-grade dysplasia in 4 cases (4/128; 3%), and equivocal in 6 cases (6/128; 5%). To further validate the consensus diagnosis, the discordant cases were reviewed by an independent expert pathologist from the United Kingdom. A total of 39 cases were reviewed blindly including the 20 cases with a revised diagnosis and 19 control cases where the Dutch expert panel agreed with the original reporting pathologists diagnosis. In 19 of the 20 cases with a revised diagnosis the British pathologist agreed that T1 colorectal cancer could not be confirmed. Additionally, amongst the 19 control cases the British pathologist was unable to confirm T1 colorectal cancer in a further 4 cases and was equivocal in 3 cases. In conclusion, both generalist and expert pathologists experience diagnostic difficulty distinguishing pseudo-invasion and high-grade dysplasia from T1 colorectal cancer. In order to prevent overtreatment, review of the histology of pedunculated T1 colorectal cancers by a second pathologist should be considered with discussion of these cases at a multidisciplinary meeting.

Diagnostic Challenges Caused by Endoscopic Biopsy of Colonic Polyps: A Systematic Evaluation of Epithelial Misplacement With Review of Problematic Polyps From the Bowel Cancer Screening Program, United Kingdom.

Endoscopic mucosal biopsy may misplace mucosal elements into the submucosa of colonic adenomas, mimicking invasive adenocarcinoma. Biopsy-related misplacement can be more challenging to recognize than typical misplaced epithelium (pseudoinvasion) in pedunculated polyps. We compared the features of 16 polyps with biopsy-related misplaced epithelium with those of 10 adenomas with pseudoinvasion and 10 adenomas with invasive adenocarcinoma and performed Ki67 and p53 immunostaining on all cases. Features of misplaced epithelium in polyps referred to the Bowel Cancer Screening Program Expert Board in the United Kingdom were also evaluated for the same morphologic features. Biopsy-related epithelial misplacement occurred in adenomas throughout the colon and often appeared infiltrative (69%), including epithelial cells singly dispersed within reactive fibroinflammatory stroma or granulation tissue (44%). Misplaced epithelium displayed only low-grade cytologic features and was associated with extruded mucin (75%), tattoo pigment (63%), and misplaced normal glands (38%); scant lamina propria and muscularis mucosae were often present (88% and 44%, respectively). Cases referred to the Bowel Cancer Screening Program Expert Board also contained infiltrative-appearing misplaced epithelium (91%) that was cytologically low grade (72%), contained nondysplastic glands (11%), and showed other signs of injury. In contrast, misplaced epithelium in pedunculated polyps always had a lobular contour with a rim of lamina propria, hemorrhage, and/or hemosiderin. Invasive carcinomas showed malignant cytology and desmoplasia; most (70%) lacked features of trauma. Ki67 and p53 staining was patchy and weak in the misplaced epithelium, whereas invasive carcinomas showed increased staining for one or both markers. Pathologists should be aware that endoscopically manipulated adenomas may contain misplaced epithelium that simulates malignancy.

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.

Filiform Polyposis Secondary to Colonic Tuberculosis Presenting as Acute Colo-Colonic Intussusception.

Filiform polyposis represents a rare but recognised manifestation on the varied spectrum of histopathology in colonic tuberculosis. We report a case of filiform polyposis secondary to colonic tuberculosis presenting as colo-colonic intussusception diagnosed on an abdominal computed tomography (CT) scan. The patient required urgent hemicolectomy and defunctioning ileostomy. Examination of the resected bowel lesions revealed filiform polyposis. Induced sputum samples from the patient grew Mycobacterium tuberculosis. The patient recovered well from the surgery and received treatment for tuberculosis. At last follow-up, he was awaiting the reversal of his ileostomy. The protean nature of histological findings in colonic tuberculosis and other current diagnostic challenges are discussed. The importance of maintaining a high index of suspicion for colonic tuberculosis and instituting early treatment is highlighted in this case.

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.

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.

Population-based study reveals new risk-stratification biomarker panel for Barrett's esophagus.

BACKGROUND & AIMS: The risk of progression of Barrett's esophagus (BE) to esophageal adenocarcinoma (EAC) is low and difficult to calculate. Accurate tools to determine risk are needed to optimize surveillance and intervention. We assessed the ability of candidate biomarkers to predict which cases of BE will progress to EAC or high-grade dysplasia and identified those that can be measured in formalin-fixed tissues. METHODS: We analyzed data from a nested case-control study performed using the population-based Northern Ireland BE Register (1993-2005). Cases who progressed to EAC (n = 89) or high-grade dysplasia ≥ 6 months after diagnosis with BE were matched to controls (nonprogressors, n = 291), for age, sex, and year of BE diagnosis. Established biomarkers (abnormal DNA content, p53, and cyclin A expression) and new biomarkers (levels of sialyl Lewis(a), Lewis(x), and Aspergillus oryzae lectin [AOL] and binding of wheat germ agglutinin) were assessed in paraffin-embedded tissue samples from patients with a first diagnosis of BE. Conditional logistic regression analysis was applied to assess odds of progression for patients with dysplastic and nondysplastic BE, based on biomarker status. RESULTS: Low-grade dysplasia and all biomarkers tested, other than Lewis(x), were associated with risk of EAC or high-grade dysplasia. In backward selection, a panel comprising low-grade dysplasia, abnormal DNA ploidy, and AOL most accurately identified progressors and nonprogressors. The adjusted odds ratio for progression of patients with BE with low-grade dysplasia was 3.74 (95% confidence interval, 2.43-5.79) for each additional biomarker and the risk increased by 2.99 for each additional factor (95% confidence interval, 1.72-5.20) in patients without dysplasia. CONCLUSIONS: Low-grade dysplasia, abnormal DNA ploidy, and AOL can be used to identify patients with BE most likely to develop EAC or high-grade dysplasia.

Cancer chromosomal instability: therapeutic and diagnostic challenges.

Chromosomal instability (CIN)-which is a high rate of loss or gain of whole or parts of chromosomes-is a characteristic of most human cancers and a cause of tumour aneuploidy and intra-tumour heterogeneity. CIN is associated with poor patient outcome and drug resistance, which could be mediated by evolutionary adaptation fostered by intra-tumour heterogeneity. In this review, we discuss the clinical consequences of CIN and the challenges inherent to its measurement in tumour specimens. The relationship between CIN and prognosis supports assessment of CIN status in the clinical setting and suggests that stratifying tumours according to levels of CIN could facilitate clinical risk assessment.

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.

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.

Observer agreement in the diagnosis of serrated polyps of the large bowel.

AIMS: To assess observer agreement in the diagnosis of colorectal serrated polyps, in particular, serrated adenomas and admixed polyps (i.e. 'polyps with admixed hyperplastic and adenomatous glands'). METHODS AND RESULTS: Sixty cases of large bowel polyps were assessed by four specialist gastrointestinal histopathologists and allocated into one of five categories: serrated adenoma, hyperplastic polyp, conventional adenoma, admixed polyp, and other polyps with serration. Complete agreement amongst all four assessors was seen with only two-fifths of the cases. The overall kappa value for all the assessors distinguishing between all five categories was 0.49. The kappa values for diagnosing serrated adenoma versus all other polyps, and admixed polyp versus all other polyps were 0.38 and 0.3, respectively. CONCLUSIONS: Specialist gastrointestinal histopathologists show only moderate concordance when distinguishing between serrated colorectal polyps. There is only fair interobserver agreement in the diagnosis of serrated adenomas and admixed polyps of the large bowel.

Clonality, founder mutations, and field cancerization in human ulcerative colitis-associated neoplasia.

BACKGROUND & AIMS: The clonality of colitis-associated neoplasia has not been fully determined. One previous report showed polyclonal origins with subsequent monoclonal outgrowth. We aimed to assess the clonality and mutation burden of individual crypts in colitis-associated neoplasias to try to identify gatekeeping founder mutations, and explore the clonality of synchronous lesions to look for field effects. METHODS: Individual crypts (range, 8-21 crypts) were microdissected from across 17 lesions from 10 patients. Individual crypt adenomatous polyposis coli (APC), p53, K-RAS, and 17p loss of heterozygosity mutation burden was established using polymerase chain reaction and sequencing analysis. Serial sections underwent immunostaining for p53, beta-catenin, and image cytometry to detect aneuploidy. RESULTS: In most lesions an oncogenic mutation could be identified in all crypts across the lesion showing monoclonality. This founder mutation was a p53 lesion in the majority of neoplasms but 4 tumors had an initiating K-RAS mutation. Some nondysplastic crypts surrounding areas of dysplasia were found to contain clonal p53 mutations and in one case 3 clonal tumors arose from a patch of nondysplastic crypts containing a K-RAS mutation. CONCLUSIONS: This study used mutation burden analysis of individual crypts across colitis-associated neoplasms to show lesion monoclonality. This study confirmed p53 mutation as initiating mutation in the majority of lesions, but also identified K-RAS activation as an alternative gatekeeping mutation. Local and segmental field cancerization was found by showing pro-oncogenic mutations in nondysplastic crypts surrounding neoplasms, although field changes are unlikely to involve the entire colon because widely separated tumors were genetically distinct.