1. Volume 138, Issue 4, Pages 1441-1454.e7 (April 2010)
Clonality Assessment and Clonal Ordering of Individual Neoplastic Crypts Shows Polyclonality of Colorectal Adenomas 
Christina Thirlwell, Olivia C.C. Will, E. Domingo, Trevor A. Graham, Stuart A.C. McDonald, Dahmane Oukrif, Rosemary Jeffrey, Maggie Gorman, Manuel Rodriguez–Justo, Joanne Chin–Aleong, Sue K. Clark, Marco R. Novelli, Janusz A. Jankowski, Nicholas A. Wright, Ian P.M. Tomlinson, Simon J. Leedham 
Gastroenterology 
Volume 138, Issue 4, Pages e7 (April 2010)
DOI: /j.gastro
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2. Figure 1
Polyclonality in an XO/XY patient using chromosomal and genetic markers. (A) i and ii, H&E of microadenoma with enlargement. iii, Fluorescence in situ hybridization for nuclei (4′,6-diamidino-2-phenylindole [dapi], blue), X-chromosome (fluorescein isothiocyanate, green), and Y-chromosome (CY-3, red) showing XO and XY crypts in the same adenoma. iv and v, Enlargements of single crypts. (B) XO/XY patient microadenoma with somatic mutation analysis. i, GENOTYPER analysis of amplicon around 1309 germline mutation from control lamina propria tissue. Germline (gl) allele is 5 base pairs shorter than the somatic (som) allele as a consequence of the codon 1309 five–base pair deletion mutation. ii and iii, Laser dissection of phenotypically dysplastic individual crypts showing somatic allelic loss in crypts 1–3 but no LOH in crypt 4 showing somatic mutation heterogeneity.
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3. Figure 2
Polyclonal FAP polyp with distinct APC mutations. Colectomy specimen showing solitary polyp. Microdissection of individual crypts circumferentially from around the polyp and somatic APC mutation sequencing reveals 2 clones with independent APC mutations enveloping an entrapped genotypically wild-type crypt.
Gastroenterology  , e7DOI: ( /j.gastro )
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4. Figure 3
Partially dysplastic crypts show top-down growth. (A) i, H&E stain of whole lesion (20×). ii, Higher magnification of partially dysplastic crypt to show orientation with respect to the lumen (100×). (B) i, H&E stain showing dysplastic upper half of the crypt with histologically normal lower half (250×). ii, β-catenin staining shows heavy nuclear staining in the dysplastic upper half with normal membranous pattern of staining in the lower half. iii, MIB-1 (Ki-67) antibody staining shows cells undergoing aberrant proliferation in the top half of the crypt, a process normally restricted to the bottom third of the crypt. (C) The cells in the dysplastic upper half of the crypt had an APC mutation not found in the lower half of the crypt.
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5. Figure 4
Topographic clonal map and phylogenetic tree for lesion 1. (A) This lesion was divided phenotypically into i, normal; ii, low-grade dysplasia (lgd); iii, invasive carcinoma; and iv, high-grade dysplasia (hgd). Individual crypts were dissected from across each phenotypic area. (B) Topographic clonal map. The number of dissected crypts taken from each phenotypically characterized area is listed in the top row of each table. Mutations are depicted by colored bars. When the same mutation was seen in all crypts from a subpopulation, the area was colored in. The maps provide accurate spatial orientation of crypt mutation burden because crypts were dissected from left to right across the lesion. (C) Phylogenetic tree. Tree branches represent new subclones expanding from within the existing mutant population. Cumulative mutations are represented by the different colors. The variable line thickness of the branches relates to the size of the crypt population carrying the new mutation, with minority clones represented by thinner lines. This large polyp with a foci of cancer had a gate-keeping APC mutation presumably arising in a single crypt (red x). p53 point mutation was seen in a large area of adenomatous crypts from which the cancer had arisen. 18q LOH had occurred after p53 mutation because different alleles were lost in the malignant and adenoma populations. In this case, different KRAS clones were detectable with distinct p.G12V subclones in the dysplastic tissue and the cancer arising from a p.G13D subclone. mss, microsatellite stable.
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6. Figure 5
Environmental influence and mechanistic models behind adenoma polyclonality. The transition from normal epithelium to polyclonal adenoma is depicted by the grey boxes. Different epithelial APC mutations are represented by the yellow and blue crypts. Environmental influences (green box) including background genetics and regional factors can select for mutations during adenoma initiation and progression. Mechanistic theories behind polyclonality development are summarized in the lilac box.
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7. Supplementary Figure 1
Mathematic assessment of the hypothesis that polyclonal adenomas arise through random collision. P values for the observed data are plotted as a function of the rate of second APC hits (per allele per year). For reasonable estimates of the second hit rate (<1e-5) the observed frequency of polyclonal adenomas was very unlikely to be explained by random collision. Top panel: FAP case. Bottom panel: sporadic case.
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8. Supplementary Figure 2
Individual phylogenetic trees for carcinoma-in-adenoma samples 2–11 (see Table 2.) All trees are interpreted from the experimental data. The epithelial initiating mutational event is depicted by a red cross. Tree branches represent new subclones expanding from within the existing mutant population. Cumulative mutations are represented by different colored lines. When a clonal order could not be established between 2 or more mutations, because all mutations were present in all the analyzed crypts, a multicolored line was used. Different mutations are represented by different colors as follows: truncating APC mutation, light red; 5q LOH, dark red; p53 point mutation, light yellow; 17p LOH, orange; 18q LOH A1, light grey; 18q LOH A2, dark grey; K-RAS, blue; aneuploidy, green; and MSI, brown. Individual mutations are shown in colored text according to standard nomenclature. The variable line thickness of the branches is related to the size of the analyzed crypt population carrying the mutation, with minority clones represented by thinner lines. Phenotypic appearance of the clones is shown by black text.
Gastroenterology  , e7DOI: ( /j.gastro )
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